Method for microbioligical introduction of oxygen into heterocyclic nitrogen compounds

ABSTRACT

A METHOD FOR THE MOCROBIOLOGICAL INTRODUCTION OF OXYGEN INTO THE HETEROCYCLIC RING OF N-ACYL DERIVATIVES OF POLYMETHYLENEIMINES (AZACYLOZLKANES) AND AZABICYCLOALKANES USING SPOROTRICHUM SULFURESCENS TO OBTAIN THE CORRESPONDING OXYGENATED PRODUCTS.

United States Patent Omcc 3,556,943 Patented Jan. 19, 1971 ABSTRACT OFTHE DISCLOSURE A method for the microbiological introduction of oxygeninto the heterocyclic ring of N-acyl derivatives of polymethyleneimines(azacycloalkanes) and azabicycloalkanes using Sporotrichum sulfurescensto obtain the corresponding oxygenated products.

CROSS REFERENCE This application is a continuation-in-part of copendingapplication Ser. No. 453,204, filed May 4, 1965 and now abandoned.

BACKGROUND OF THE INVENTION This invention relates to a biconversionprocess to obtain oxygenated polymethyleneimines and azabicycloalkanes,some of which are known but which have not hitherto been readilyavailable by purely chemical methods of synthesis.

SUMMARY OF THE INVENTION This invention relates to a novel method forthe introduction of oxygen into the heterocyclic ring of N-acylderivatives of polymethyleneimines (azacycloalkanes) andazabicycloalkanes. More particularly this invention relates to theintroduction of oxygen into the 'meterocyclic ring of N-aromatic acylderivatives of polymethyleneimines (azacycloal'kanes) andazabicycloalkanes by subjecting them to the oxygenating activity of themicroorganism Sporotn'chum sulfurescens, to certain novel productsproduced by the process of this invention and to derivatives thereof.

DETAILED DESCRIPTION OF THE INVENTION The process of this invention isof particular value for the introduction of oxygen into the heterocyclicring of compounds having the formula:

wherein n is a whole number from 4 to 12, inclusive, X and X are eachselected from the group consisting of hydrogen, methyl, ethyl, andpropyl, and X and X taken together constitute a bridge containing from 1to 3 carbon atoms, inclusive, and Z is selected from the groupconsisting of in which R is aryl, and R is aralkyl.

The above compounds of Formula I when subjected to the bioconversionprocess of this invention give rise to the corresponding compoundswherein the heterocyolic ring has been oxygenated by the introduction ofa hydroxy or keto group as represented by Formula Ia.

wherein X, X, and Z have the meanings previously given and Y is hydroxyor keto.

The bioconversion process of this invention when applied to thecompounds of Formula I, which contain one or more attached alkyl groups,namely, the compounds of Formula I wherein X and X are methyl, ethyl orpropyl also give rise to the corresponding compounds wherein the alkylside chain has been oxygenated by the introduction of a hydroxy or ketogroup. These compounds are likewise useful for the samepurposes ashereinafter given for the compounds in which the heterocyclic ring hasbeen oxygenated.

The bioconversion process of this invention is of particular value forthe production of compounds having the formula:

wherein X, X, and Z have the meanings previously given, n is a wholenumber from 1 to 5, inclusive, and n is a whole number from 2 to 8,inclusive, in which the sum of m and n is not less than 3 and not'morethan 11.

The compounds produced by the bioconversion process of this invention,wherein the introduced oxygen substituent is hydroxy, for example thecompounds of Formula II, can be chemically oxidized by methodshereinafter disclosed, for example using chromic acid, to obtain thecorresponding keto compounds, particularly those having the formula:

wherein n n X, X, and Z have the meanings and limitations previouslygiven.

The compounds produced by the bioconversion process wherein theintroduced oxygen substituent is keto, or the keto compounds produced bythe above chemical oxidation step, can if desired be reduced by methodshereinafter disclosed, for example using sodium borohydride, to obtainthe corresponding hydroxy compounds.

While the process of the invention is of general applicability and canbe employed for the oxygenation of all of the N-acyl derivatives ofpolymethyleneimines and azabicycloalkanes of Formula I, above, to obtainthe oxygenated compounds of Formulae II and III, above, the process ofthe invention is especially advantageous for obtaining compounds of theinvention as represented by Formulae IV, V, VI and VII below:

(OHQM wherein n is a whole number from 1 to 3, inclusive, n is a wholenumber from 3 to 5, inclusive, and n is a whole number from 6 to 8,inclusive, in which the sum of n and n is not less than 5 and notgreater than 7 and the sum of n, and n is 11, n, is 0 or 1 and n is 0 0r1, in which n and 12', cannot each equal 0 simultaneously; W is aradical selected from the group consisting of carbonyl,hydroxymethylene, acyloxymethylene, methylhydroxyme'thylene,phenylhydroxymethylene, alkylenedioxymethylene, andcyclicaminomethylene; Z is selected from the group consisting ofhydrogen, cycloalkyl, cyanomethyl, Z-aminoethyl, Z-guanidinoethyl,

in which R is aryl and R is aralkyl, and Y is hydrogen, halogen, hydroxyor alkoxy.

In this application the term aryl means an aryl radical of 6 to 12carbon atoms, inclusive, such as, for example, phenyl, tolyl, xylyl,naphthyl, biphenylyl and the like. The term aralkyl means an aralkylradical of 7 to 16 carbon atoms, inclusive, such as benzyl,p-nitrobenzyl, 4-methylbenzyl, S-methylbenzyl, 4-methylphenethyl,4-biphenylbutyl, ot-naphthylmethyl, Bnaphthylethyl, and the like. Theterm acyloxymethylene means a substituent wherein the acyl radical isthat of an organic carboxylic acid of from 1 to 16 carbon atoms,inclusive, such as those hereinafter defined. The term"alkylenedioxymethylene means the radical in which Q is a lower alkyleneradical containing from 2 to 8 carbon atoms, inclusive, and having from2 to 3 carbon atoms, inclusive, in th chain connecting the in which thesymbol Av is a saturated 5 to 9 ring atom cyclic amino radical such asthose hereinafter listed. The term cycloalkyl means a cycloalkyl radicalof 5 to 15 carbon atoms inclusive, such as cyclopentyl, cyclohexyl,cyclooctyl, cyclodecyl, cyclopentadecyl and the like. The term alkoxymeans an alkoxy radical wherein alkyl is an alkyl radical of 1 to 6carbon atoms.

The oxygenated compounds produced by the bioconversion and oxidationprocesses of this invention and the derivatives thereof, including thoserepresented by Formulae II, III, IV, V, VI and VII, above; and thecompounds produced in the examples appended hereto are useful asinsecticides, fungicides, parasiticides, protein denaturants, insectrepella-nts, high boiling solvents, plasticizers for synthetic resins,crosslinking agents for fiber synthesis, pharmacologic agents forpsychic control effects and as intermediates for dyes, polymers andfibers.

As an example of their use as intermediates the compounds wherein theoxygen substituent is keto (the hydroxy compounds can be oxidized toketo) can be converted to lactams which can by hydrolyzed to amino acidsin accordance with the procedures disclosed in US. Pats. 2,579,851 and2,569,114. For example, the ketones are converted to the oximes byreacting them with hydroxylamine or a salt thereof. The oximes are thensubjected to a Beckman rearrangement by treatment with sulfuric acid orthe equivalent to produce lactams. The lactams thus produced are usefulintermediates giving on hydrolysis amino acids. The lactams and aminoacids thus obtained are useful for the manufacture of valuable products,for example, polyamides,

, as disclosed in US. Pat. 2,579,851, supra.

For use as insect repellents 'the compounds of this invention can beformulated with aqueous or nonaqueous carriers in accordance withmethods known in the art, for example, US. Pat. 3,131,215.

The microbiological process of this invention comprises subjecting anN-aromatic acyl derivative of a methyleneimine (I) to the oxygenatingactivity of the species of fungus Sporotrichum sulfurescens. The genusSporotrichum belongs to the family Moniliaceae of the order Monilialesof the class Deuteromycetes.

The typical strain preferred for the practice of this invention isSporotrichum sulfurescens, available from the American Type CultureCollection, Washington, DC, Collection No. ATCC 7159. It is to beunderstood, however, that other strains of Sprootrichum sulfurescens aresuitable for the practice of this invention.

The starting materials (I) for the process of this invention, some ofwhich are known, are prepared from polymethyleneimines (azacycloalkanes)and azabicycloalkanes of the formula:

H (mai N wherein n, X and X have the meanings previously given, bymethods known in the art for acylating polymethylenea imines. Forexample, the selected polymethyleneimine or azabicycloalkane isdissolved, mixed or suspended in an aqueous sodium hydroxide solutionand reacted with the acid halide of the appropriate mouobasic aryl oraralkyl carboxylic acid, such as those, hereinafter listed as acylatingagents, or with the acid halide of the appropriate monobasic arylsulfonic acid such as, for example, benzenesulfonic acid, m-,p-toluenesulfonic acids, orand ,B-naphthalene sulfonic acids,p-chlorobenzenesulfonic acid and the like, as illustrated byPreparations 1 through 10, herein. The acylation may also be carried outin hydrocarbon solvents such as, e.g., Skellysolve B (mixed hexanes),benzene or toluene. In carrying out the bioconversion process of thisinvention the operational conditions and reaction procedures areadvantageously those known in the art of bioconversion as illustrated inMurray et al., US. Pats. 2,602,769 and 2,735,800, utilizing theoxygenating activity of the microorganism Sporotrichum sulfurescens.

In the practice of this invention, the bioconversion can be effected bya growing or resting culture of the microorganism or by spores, washedcells or enzymes of the microorganism.

Culture of the microorganism for the purpose and practice of thisinvention is in or on a medium favorable to development of themicroorganism. Sources of nitrogen and carbon should be present in theculture medium and an adequate sterile air supply should be maintainedduring the conversion, for example, by the conventional techniques ofexposing a large surface of the medium or by passing air through asubmerged culture.

Nitrogen in assimilable form can be provided by sources normallyemployed in such processes, such as corn steep liquor, soybean meal,yeast extracts, peptone, soluble or insoluble vegetable or animalprotein, lactalbumin, casein, whey, distillers solubles, amino acids,nitrates and ammonium compounds, such as ammonium tartrate, nitrate,sulfate and the like.

Available carbon can also be provided by sources normally used inbioconversions such as carbohydrates, e.g., glucose, fructose, sucrose,lactose, maltose, dextrines, starches; meat extracts, peptones, aminoacids, proteins, fatty acids, glycerol, whey and the like. Thesematerials may be used either in a purified state or as concentrates suchas whey concentrate, corn steep liquor, grain mashes, and the like, oras mixtures of the above. Many of the above sources of carbon can alsoserve as a source of nitrogen.

The medium can desirably have a pH before inoculation of between about 4to about 7 through a higher or lower pH can be used. A temperaturebetween about 25 to 32 C. is preferred for growth of the microorganismbut higher or lower temperatures within a relatively wide range aresuitable.

The substrate can be added to the culture during the growth period ofthe microorganism as a single feed or by gradual addition during theconversion period or it can be added to the medium before or aftersterilization or inoculation making appropriate adjustments for effectsof pH and/or temperature upon the stability of the substrate used. Thepreferred, but not limiting, range of concentration of the substrate inthe culture medium is about 0.1 to 10 grams per liter. The substrate isadded to the medium in any suitable manner, especially one whichpromotes a large surface contact of the substrate to the oxidizingactivity of the microorganism, for example, by dissolving the substrate,when it is a solid, in an organic solvent and mixing the solutionthoroughly with the medium or by adding to the medium finely comminutedparticles of the substrate, e.g., micronized particles, preferably 90%by weight smaller than 20 microns, either as a dry powder or, preferablyfor mechanical reasons, as an aqueous suspension. In preparing theaqueous suspension, the use of dispersing or suspending agents isadvantageous.

The temperature during the fermentation can be the same as that foundsuitable for growth of the microorganism. It need be maintained onlyWithin such range as supports life, active growth or the enzyme activityof the microorganism; the range of 20 to 35 C. is preferred. A pH ofabout 4 to 6 is generally preferred for growth of the microorganismduring the bioconversion but for acidsensitive substrates, the pH shouldbe about 7 during the fermentation. Aeration can be effected by surfaceculture or preferably under submerged fermentation conditions, inaccordance with methods well known in the art. The time required foroxygenation by the enzymatic system of the microorganism employed canvary considerably. Therange of about 2 to hours is practical but notlimiting; 72 hours is generally satisfactory. The progress of thebioconversion and its completion are conveniently determined bypaper-strip chromatography, vapor-phase chromatography or thin-filmchromatography [Heftman, Chromatography (1961), Reinhold Publishing Co.,New York, N.Y.].

Alternatively, the oxygenation of the selected substrate can be effectedunder aerobic conditions by subjecting it to the oxygenating action ofoxygenating enzymes isolated from the microorganism, to the action ofspores of the microorganism, and to the action of isolated cells of themicroorganism. Isolated enzyme preparations can be prepared inaccordance with the general procedure disclosed by Zuidweg et al.,Biochim. Biophys. Acta, 58, 131-133 (1962). Oxygenation can be effectedwith spores in accordance with the general process disclosed in US.Pats. 3,031,379 and 3,031,382. The separation of washed cells from thefermentation medium is well known in the art, see for example US. Patent2,831,789.

The term oxygenating activity as used throughout this specificationmeans the enzymatic action of a growing or resting culture of themicroorganism or of spores, washed cells or isolated enzymes of themicroorganism, which effects introduction of oxygen in the molecule ofthe substrate, under aerobic fermentation conditions.

After completion of the fermentation, the resulting oxygenated productsare recovered from the fermentation beer by conventional methods. Forexample, the Whole beer can be extracted with a water-immiscible organicsolvent such as methylene chloride, chloroform,

carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amylacetate, benzene, and the like or the beer and mycelia can be separatedby conventional methods such as centrifugation or filtration, and thenseparately extracted with suitable solvents. The mycelia can beextracted with either water-miscible or water-immiscible solvents or incases where little or no product is contained in the mycelium, it can bemerely washed with water and the wash water added to the beer filtrate.The beer, free of mycelia, can then be extracted with waterimmisciblesolvents such as those listed above. The extracts are combined, driedover a drying agent such as anhydrous sodium sulfate, and the solventremoved by conventional methods such as evaporation or distillation atatmospheric or reduced pressure. The oxygenated products thus obtainedcan be further purified by conventional methods, e.g.,recrystallization, chromatography, distillation in the case of liquids,and the like.

Separation of the various oxygenated products obtained from thefermentation can be accomplished by conventional methods such aschromatography and/or fractional crystallization and, if liquids, bydistillation. 'In instances when separation of the hydroxy compounds isdiificult a convenient and advantageous method is first to oxidize underacidic neutral or slightly basic conditions the crude oxygenatedpolymethyleneimines or azabicycloalkanes obtained from the beer inaccordance with methods known in the art for oxidizing secondary hydroxygroups to ketones, for example, Fieser and Fieser, Natural ProductsRelated to Phenanthrene, 3rd ed., pp. 127-129, 193 and 194, RheinholdPublishing Corporation, New York, NY. Thus, the crude bioconversionproducts are dissolved in an inert organic solvent such as acetone,benzene, methylene chloride, t-butanol, and the like, and then oxidizedwith aqueous chromic acid, potassium permanganate, t-butyl hypochlorideor like oxidizing agents to convert the secondary hydroxy groups presentto keto, thereby producing a mixture of the corresponding keto compoundswhich are in some cases more easily separated by chromatography and/orcrystallization or distillation in the case of liquids.

The compounds obtained from the bioconversion wherein the introducedoxygen substitutent is keto or those prepared by the above chemicaloxidation of the corresponding hydroxy compounds, for example, compoundsof Formula III, can, if desired, be reduced, preferably under neutral oracidic conditions, in accordance with methods known in the art forreducing carbonyl groups to produce the corresponding hydroxy compounds.For example, reduction can be conveniently accomplished with hydrogen inthe presence of a atalyst such as palladium, platinum or R'aney nickelunder neutral conditions; sodium in an alkanol; or with a reducing agentsuch as lithium aluminum hydride, sodium borohydride, primary isobutylmagnesium bromide or lithium tritertiary butoxy aluminum hydride, andthe like.

The compounds obtained from the bioconversion wherein the introducedoxygen substituent is hydroxy, for example the compounds of Formula II,can be acyl-ated to give the corresponding acyloxy compounds inaccordance with methods known in the art for acylating secondary hydroxygroups, for example, by reaction with the appropriate acid anhydride oracid halide, by reaction with the appropriate ester or by reaction withthe appropriate acid in the presence of an esterification catalyst, etc.Suitable acylating agents are organic carboxylic acids, particularlyhydrocarbon carboxylic acids containing from 1 to 16 carbon atoms,inclusive, or acid anhydrides or acid halides thereof. Illustrative ofhydrocarbon carboxylic acids employed in the formation of the acylatesare saturated and unsaturated aliphatic acids and aromatic aids such asacetic, porpionic, butyric, isobutyric, tert.-butylacetic, valeric,isovaleric, caproic, caprylic, decanoic, dodecanoic, acrylic, crotonic,hexynoic, heptynoic, octynoic, cyclobutanecarboxylic,cyclopentanecarboxylic, cyclopentenecarboxylic, oyclohexanecarboxylic,dimethylcyclohexanecarboxylic, benzoic, toluic, naphthoic, ethylbenzoic,phenylacetic, naphthaleneacetic, phenylvaleric, cinnamic,phenylpropiolic, phenylpropionic, p-butoxyphenylpropionic, succinic,glutaric, dimethylglutaric, maleic, cyclopentylpropionic acids, and thelike. If the acylating agent is free acid, the reaction is preferablyeffected in the presence of an esterification catalyst, for examplep-toluenesulfonyl chloride, trifluoroacetic anhydride, p-toluenesulfonicacid, trifluoroacetic acid, sulfuric acid, and the like.

The compounds obtained from the bioconversion process wherein theintroduced oxygen substitutent is keto or the keto compounds obtained bychemical oxidation of the corresponding hydroxy compounds such as thecompounds of Formula III, can be converted to their common carbonylderivatives such as oximes, hydrazones, semicarbazones, cyclic alkyleneketals and the like in accordance with methods well known in the art.For example, the carbonyl group can be ketalized by reacting theselected compound with an alkanediol selected from the group of vicinalalkane-l,2-diols and alkane-l,3- diols containing up to and including 8carbon atoms, e.g., ethylene, propylene, trimethylene, 2,3-butylene,2,4- pentylene, 4-methyl-l,2-pentylene, 1,3-hexylene, 1,2- heptylene,3,4-heptylene, 1,3-octylene, and the like, preferably in an organicsolvent such as benzene, toluene, xylene, methylene chloride, and thelike and in the presence of an acid catalyst such as p-toluenesulfonicacid. The reaction is conducted at a temperature between about 20 andabout 200 0., preferably between about 40 and and about C. The timerequired for the reaction is not critical and may be varied betweenabout 1 and 48 hours, depending on the temperature.

The acylates and the carbonyl derivatives can, if de sired, be removedby hydrolysis in accordance with methods known in the art, e.g., withdilute acids or bases. Other derivatives of the oxygenated bioconversionproducts of the invention can be prepared in accordance with methodsknown in the art; for example, the compounds which have the radical,

o H OR attached to the nitrogen can be reduced using lithium aluminumhydride or diborane to obtain the corresponding aralkyl amines havingthe partial formula,

If additional unprotected carbonyl groups are present in the moleculethey will be concomitantly reduced to hydroxymethylene, and can beoxidized back to carbonyl, if desired, using chromic acid (Jonesreagent) as hereinbefore described. The aralkyl amines thus obtained canbe hydrogenolyzed, e.g. using hydrogen in the presence of a catalyst,such as palladium on carbon or Raney nickel to give the correspondingsecondary amine, NH. The secondary amine, thus obtained can be reactedwith chloroacetonitrile in the presence of sodium carbonate to obtainthe corresponding cyanomethylamine, which is reduced with lithiumaluminum hydried to the corresponding Z-aminoethyl derivative having thepartial formulas, NCH CH NH The aminoethyl compound thus obtained isthen reacted with 2-methyl-2- thiopseudourea sulfate to obtain thecorresponding 2- guanidinoethylamine sulfate having the partial formula:

which can be neutralized with alkali to obtain the corresponding freebase. The secondary amaines obtained, above can be cycloalkylated at thenitrogen group by reacting with a cycloalkanone followed by reductionwith lithium aluminum hydride to obtain the corresponding cycloalkylamines.

The compounds of the invention wherein the substituent Z or Z is whereinR and R have the meaning previously given, and which have a hydroxygroup attached to the heterocyclic ring, can be subjected to acylmigration, e.g. with hydrochloric acid in tetrahydrofuran, which resultsin acylation of the hydroxy group to obtain the corresponding secondaryamine.

The compounds of this invention, wherein the substituent Z or Z is:

in which R is' aryl and R is aralkyl and which have a keto groupattached to the reterocyclic ring, can be halogenated on theheterocyclic ring and then subjected to solvolysis with an alcohol inthe presence of sodium hydroxide to obtain the corresponding alkyloxycom pound. The compounds of this invention having the substituentsdescribed above can also be reacted with a Grignard reagent, RMgX,wherein R is alkyl or aryl, as hereinbefore defined and X is halogen, inaccordance with known methods to obtain the corresponding compoundswherein the keto substituent is replaced by bydroxy and the R radical ofthe particular Grignard reagent employed. The acyl substituent on thenitrogen is replaced by hydrogen during the Grignard reaction.

The following preparations and examples are intended to illustrate theprocess as applied to representative and typical starting materials. Thefollowing examples are for the purpose of illustrating the best modecontemplated of carrying out the invention and to supplement theforegoing disclosure of the invention with additional descriptions ofthe manner and process of carrying out the invention so as further toenable workers skilled in the art to do so.

In addition to the uses given hereinabove, the compounds of thisinvention which are free amines form salts with fluosilicic acid whichare useful as mothproofing agents in accordance with U.S. Pat. 1,915,334and 2,075,359. These amines also form salts with thiocyanic acid whichcondense with formaldehyde to form resinous materials useful as picklinginhibitors according to US. Pat. 2,425,320 and 2,606,155.

PREPARATION 1 1 benzoylhexamethyleneimine 1- (p-toluenesulfonyl)-hexamethyleneimine A mixture of 9.92 g. of hexamethyleneimine, 100 ml.of 2 N sodium hydroxide solution, and 18 g. of ptoluenesulfonyl chloridewas shaken vigorously for fifteen minutes (heat of reaction noted) andallowed to stand for two hours. The product was recovered by filtration,washed with water, dried and then recrystallized from aqueous acetoneand gave 23.0 g. of l-(p-toluenesulfonyl)-hexamethyleneimine, M.P. 7173C.

Analysis.Calcd. for C H NO S (percent): C, 61.62; H, 7.56; S, 12.66.Found (percent): C, 61.83; H, 7.72; S, 12.94.

PREPARATION 3 1-henzoylheptamethyleneimine A mixture of 30 g. ofheptamethyleneimine, 100 m1. of 50% sodium hydroxide solution, and 540ml. of water was cooled to 15 C. Benzoyl chloride (43 g.) was addeddropwise, with vigorous stirring and with the temperature maintained at15-20" C. Stirring was continued for 1 hour after the end of theaddition and the oily product recovered by ether extraction. Evaporationof the ether afforded 55 g. of l-benzoylheptamethyleneimine as an oil.

PREPARATION 4 1-benzoyloctamethyleneimine A mixture of g. ofoctamethyleneimine, 20 ml. of 50% sodium hydroxide and 100 ml. of water,was cooled to about C. and 12.5 g. of benzoyl chloride was addeddropwise with vigorous stirring keeping the temperature 1520 C. Stirringwas continued for about 1 hour after the end of the benzoyl chlorideaddition, and the product was recovered by methylene chlorideextraction. Evaporation of the solvent gave 17.6 g. of1-benzoyloctamethyleneimine as an oil.

PREPARATION 5 l-benzoylalkylpiperidines Following the procedure ofPreparation 4, above, 2- propylpiperidine was converted to1-benz0yl-2-propylpiperidine; 4 propylpiperidine to 1 benzoyl 4propylpiperidine; 2 methyl 5 ethylpiperidine to 1 benzoyl- 2 methyl 5ethylpiperidine; 2,6 dimethylpiperidine to 1 benzoyl 2,6dimethylpiperidine; 2 methylpiperidine to l-benzoyl 2 methylpiperidine;3 methylpiperidine to l-benzoyl 3 methylpiperidine; 4 methylpiperidineto 1 benzoyl 4 methylpiperidine; 2 ethylpiperidine to 1 benzoyl 2ethylpiperidine and 3- methyl 3 phenylpiperidine to 1 benzoyl 3 methyl-3-phenylpiperidine.

PREPARATION 6 1-benzoyldodecarnethyleneimine Azacyclotridecane-Z-one (40g. in a Soxhlet thimble) was continuously extracted into a refluxingsolution of 15 g. of lithium aluminum hydride in one liter of ether. Thereaction was allowed to continue four hours after all of the amide hadbeen added. Water was added to destroy the excess hydride and the etherphase was separated, filtered through sodium sulfate, and concentratedin vacuo to give 36.8 g. of oily dodecamethyleneimine. This was added to30 ml. of 50% sodium hydroxide in 170 ml. of water, cooled to 20 C. withan ice bath, and 30 g. of benzoyl chloride was added slowly withvigorous stirring. The resultant l-benzoyldodecamethyleneimineprecipitated from solution, was collected on a filter and washed withwater. Recrystallization from acetone-Skellysolve B gave 47.5 g. ofl-benzoyldodecmethyleneimine, M.P. 5l53 C.; for analysis a sample wastwice recrystallized from acetone-Skellysolve B to M.P. 5456 C.

Analysis.-Calcd. for C H NO (percent): C, 79.39; H, 10.17; N, 4.87.Found (percent): C, 79.43; H, 10.40; N, 5.03.

PREPARATION 7 Benzyl-3 -azabicyclo [3 .2.2] nonane-3 -carboxylate Asuspension of 12.5 g. of 3-azabicyclo[3.2.2]nonane in 115 ml. of 2 Nsodium hydroxide was stirred and chilled while adding 17 g. ofcarbobenzoxy chloride maintaining the temperature between 17-20 C. Themixture was stirred with no temperature control for one hour and thenextracted with ether. The extract was washed with dilute hydrochloricacid, water, dilute sodium bicarbonate, water, dried over sodiumsulfate, and the solvent removed to give a light yellow oil. The oilthus obtained was chromatographed over 500 g. of Florisil (syntheticmagnesium silicate, hereinafter called Florisil) and eluted withSkellysolve B and Skellysolve B containing increasing proportions ofacetone (2 to 6% acetone). Those fractions which by thin layerchromatography were found to contain the desired product were combinedand evaporated to give 15.80 g. of benzyl-3-azabicyclo[3.2.2]nonane-3-carboxylate as a colorless oil.

Analysis.-Calcd. for C H NO (percent): C, 74.10; H, 8.16; N, 5.40. Found(percent): C, 74.68; H, 8.22; N 5.49.

PREPARATION 8 3-(p-toluenesulfonyl)-3-azabicyclo[3.2.2]nonane Asuspension of 12.5 g. of 3-azabicyclo[3.2.2]nonane in ml. of 2 N sodiumhydroxide was treated with 19.1 g. of p-toluenesulfonyl chloride. Themixture was shaken vigorously. No heat of reaction was noted so themixture was heated at the boiling point for several minutes, and cooledto room temperature. The lumpy product thus obtained was recovered byfiltration, washed with water, and recrystallized from acetone-water;yield 21.0 g. of 3 (p toluensulfonyl)-3-azabicyclo[3.2.2]-

nonane, M.P. 115118 C.; an analytical sample from methanol melted at115-117 C.

Analysis.-Calcd. for C H NO S (percent): C, 64.48; H, 7.58; N, 5.01; S,11.48. Found (percent): C, 64.20; H, 7.76; N, 5.04; 5, 11.53.

PREPARATION 9 3-benzoyl-3-azabicyclo [3 .2.2] nonane To a vigorouslystirred mixture of 500 g. of 3-azabicyclo[3.2.2]nonane and 3200 ml. of10% sodium hydroxide solution was added 500 ml. of benzoyl chloride.After minutes an additional 250 ml. of benzoyl chloride was added andstirring was continued for three hours. The product was recovered,washed with water and dried; yield 900 g. of3-benzoyl-3-azabicyclo[3.2.2]- nonane, M.P. 9295 C.; an analyticalsample from ether-hexane melted at 93-94 C.

Analysis.-Calcd. for C H NO (percent): C, 78.56; H, 8.35; N, 6.11. Found(percent): C, 78.79; H, 8.52; N, 6.26.

PREPARATION 10 2-benzoyl-2-azabicyclo [2.2.2] octane To a stirredmixture of 50 g. of crude 2-azabicyclo- [2.2.2]octane, 100' ml. of 50%sodium hydroxide solution, and 800 ml. of ice plus water was added 50ml. of 'benzoyl chloride. Stirring was continued for 1 hour after theaddition, and the crude product recovered by filtration and washed withwater. The solid product thus obtained was taken up in methylenechloride and filtered through about 100 m1. of Florisil, washed with 1l. of methylene chloride and then 1 1. of 10% acetone- Skellysolve B.Evaporation and recrystallization from acetone-Skellysolve B gave2-benzoyl-2-azabicyclo- [2.2.2]octane, 33.8 g., M.P. 100104 C. Foranalysis a sample was thrice recrystallized from the same solvent pairto M.P. 115118 C.

Analysis.Calcd. for C H NO (percent): C, 78.10; H, 7.96; N, 6.51. Found(percent): C, 77.85; H, 7.22; N, 6.22.

EXAMPLE 1 Oxygenation of l-benzoylpiperidine A medium was prepared of200 g. of cornsteep liquor (60% solids), 100 g. of commercial dextrose,and 10 l. of tap water. The pH was adjusted to between 4.8 and 5 and 10ml. of lard oil was added as a foam preventive. This medium wassterilized and inoculated with a 72- hour vegetative growth ofSporotrichum sulfurescens, ATCC 7159, and after incubation for 24 hoursat a temperature of about 28 C. using a rate of aeration of 0.5 l. perminute and agitation at 300 r.p.m., the substrate, 2 g. ofl-benzoylpiperidine in solution in a minimum amount of acetone (aboutml.) was then added to the fermentation. After an additional 72-hourperiod of incubation at the same temperature and aeration, the beer andmycelium were separated by filtration. The mycelium was washed withwater and the wash-water was added to the beer filtrate. The thusdbtained beer filtrate was extracted four times with a volume ofmethylene chloride equal to one-fourth the volume of the filtrate. Thecombined extracts were washed with onefourth volume of distilled Waterand the solvent was removed by distillation to give a residue comprising1-benzoyl-4-hydroxypiperidine.

Chromatography of the extract residue over Florisil and elution withSkellysolve B containing increasing proportions of acetone gave 407 mg.of crude 1-benzoyl-4- hydroxypiperidine in the late acetone-SkellysolveB eluate fractions. The product was characterized by the followingderivatives.

Oxidation of 151 mg. of the crude 4-hydroxy compound with Jones chromicacid reagent gave 137 mg. of crude 1-benzoyl-4-oxopiperidine whichafiorded 89 mg. of the 2,4-dinitrophenylhydrazone, M.P. 196498 C. Theremaining crude 4-hydroxy compound was treated with 12 phenyl isocyanateto :give, after chromatography on Florisil, 89 mg. of thephenylurethane, M.P. 184.5- 186.5 C.

EXAMPLE 2 Oxygenation of l-benzoylhexamethyleneimine The bioconversionand extraction procedures of Example 1 were repeated using 2 g. ofl-benzoylhexamethyleneimine as the substrate. The residue thus obtainedwas chromatographed on Florisil and eluted with Skellysolve B containingincreasing proportions of acetone. The 25 acetone-Skellysolve B eluategave about 250 mg. of 1 benzoyl-4-oxohexamethyleneimine and the acetoneeluate gave 1-benzoyl-4-hydroxyhexamethyleneimine, determined by thinlayer chromatography.

The 1-benzoyl-4-hydroxyhexamethyleneimine thus obtained was dissolved inacetone and oxidized at room temperature by the addition of a visibleexcess of Jones reagent (2.67 M chromic acid reagent prepared from 26.7g. of chromium trioxide and 23 ml. of sulfuric acid diluted to ml. withwater). The excess oxidant was destroyed by the addition of isopropylalcohol and the mixture was evaporated to dryness. Water (2-0 ml.) wasadded, and the product was extracted with 20 ml. of methylene chloride.The extract was evaporated to dryness and the re sidual1-benzoyl-4-oxohexamethyleneimine thus obtained was combined with thesame product obtained directly from the bioconversion. The combinedproduct was chromatograp'hed on a column of Florisil. The column waseluted with Skellysolve B containing increasing proportions of acetoneand those fractions containing the desired product, determined by thinlayer chromatography were combined and evaporated to give about 770 mg.of l-benzoy1-4-oxohexamethyleneimine as an oil, B.P. 170174/ 0.3 torr,that crystallized slowly.

Analysis.Calcd. for C H NO (percent): C, 71.86; H, 6.96; N, 6.45. Found(percent): C, 71.51; H, 7.25; N, 6.46.

The 2,4-dinitrophenylhydrazone derivative of l-benzoyl-4-oxohexamethyleneimine melted at 173.5480 C.

EXAMPLE 3 Oxygenation of 1-benzoylhexamethyleneimine The bioconversion,oxidation, extraction and chromatographic procedures of Example 1 wererepeated on a larger scale using 5000 1. of sterilized medium of thesame composition and 1 kg. of l-benzoylhexamethyleneimine as thesubstrate to give 96.9 g. of l-benzoyl-4-oxohexamethyleneirnine and12.57 g. of l-benzoyl-3-oxohexamethyleneimine. The 3-oxo product wasrecrystallized three times from ethyl acetate-Skellysolve B hexanes togive l-benzoyl 3 oxohexamethyleneimine, M.P. 113-114 0.; 1 1705, 1625, vl600, 1575, 1495; 11,785, 750, 705 cm.- in Nujol.

Analysis.-Calcd. for C H NO (percent): C, 71.86; H, 6.96; N, 6.45. Found(percent): C, 71.48; H, 6.75; N, 6.49.

EXAMPLE 4 Oxygenation of l-(p-toluenesulfonyl)-hexamethyleneimine Thebioconversion and extraction procedures of Example 1 were repeated using2 g. of l-(p-toluenesulfonyD- hexamethyleneimine as the substrate. Theresidue from the beer extract thus obtained was chromatographed over 100g. of Florisil by the gradient elution method. The residue was placed onthe Florisil with 75 ml. of methylene chloride; elution was with 4 l. ofSkellysolve B containing increasing proportions of acetone from 030% andcollecting fractions of ml. each. The fractions which contained thedesired product, l-(p-toluenesulfonyl)-4-hydroxyhexamethyleneimine(determined by thin layer chromatography) were combined, evaporated toremove the solvent mixture, redissolved in acetone and oxidized withJones reagent in the same manner as disclosed in Example 2, above, togive a crude yellow solid, which was crystallized from ether to give0.33 g. l-(p-toluenesulfonyl)-4-ox0hexamethyleneimine, M.P. 81 C.

Analysis.-Calcd. for C H NO S (percent): C, 58.40; H, 6.64; N, 5.24; S,12.00. Found (percent): C, 58.40; H, 6.47; N, 5.26; S, 12.24.

Recrystallization of the l-(p-toluenesulfonyl)-4-oxohexamethyleneiminethus obtained from acetone-Skellysolve B gave a crystallinemodification, M.P. 89-90 C.

EXAMPLE 5 Oxygenation of 1-benzoylheptamethyleneimine The bioconversionand extraction procedures of Example l were repeated usingl-benzoylheptamethyleneimine as the substrate. The extract residue thusobtained, comprising a mixture of 1-benzoyl-4-hydroxyheptamethyleneimineand l-benzoyl-5-hydroxyheptamethyleneimine, was chromatographed onFlorisil. The hydroxylated 1- benzoylheptamethyleneimines were elutedwith acetone and oxidized with excess Jones chromic acid reagent to thecorresponding ketoamides, which were separated by chromatography onFlorisil and eluted with Skellysolve B containing increasing proportionsof acetone. The more polar ketoamide was eluted with 25%acetone-Skellysolve B and recrystallized from acetone-Skellysolve B togive 1. benzoyl-5-oxoheptamethyleneimine, M.P. 122-124 C.

Analysis.-Calcd. for C H NO (percent): C, 72.70; H, 7.41; N, 6.06. Found(percent): C, 72.63; H, 7.59; N, 6.31.

The less polar 1-benzoyl-4-oxoheptamethyleneimine was obtained as an oilfrom early 25% acetone-Skellysolve B eluate fractions.

EXAMPLE 6 Oxygenation of 1-benzoyldodecamethyleneimine The bioconversionand extraction procedures of Example l were repeated usingl-benzoyldodecamethyleneimine as the substrate. The extract residue thusobtained was analyzed by thin layer chromatography and found to containa mixture of hydroxy-l-benzoyldodecamethyleneimines, the majorcomponents of which were l-benzoyl-S- hydroxydodecamethyleneimine,l-benzoyl-6-hydroxydodecamethyleneimine andl-benzoyl-7-hydroxydodecamethyleneimine. The extract residue was thenchromatographed on Florisil and the mixedhydroxy-4-benzoyldodecamethyleneirnines were eluted with acetone andoxidized in acetone with excess Jones reagent in the same manner asdisclosed in Example 2, above. The resulfing mixture of ketoamides waschromatographed on Florisil. Elution with 25% acetone-Skellysolve B andthen, withacetone afforded, successively,l-benzoyl-5-oxododecamethyleneimine, 1 benzoyl 6Oxododecamethyleneimine, and lbenzoyl-7-oxododecamethyleneimine.

EXAMPLE 7 Oxygenation of 2-benzoy1-2-azabicyclo[2.2.2]octane Thebioconversion and extraction procedures of Example 1 were repeated using2 g. of 2-benzoyl-2-azabicyclo[2.2.2]octane as the substrate.

The extract residue thus obtained was chromatoa graphed on Florisil.Elution with 25% acetone-Skellysolve G gave 2benz0yl-2-azabicyclo[2.2.2]octan-S-ol, which was recrystallized fromacetone-Skellysolve B to M.P. 139-141 C.

Analysis.-Calcd. for C H NO (Percent): C, 72.70; H, 7.41; N, 6.06. Found(percent): C, 72.52; H, 7.19; N, 6.18.

The 2-benzoyl-5-hydroxy 2 azabicyclo[2.2.2]octane thus obtained isoxidized with Jones reagent in the manner disclosed in Example 2, above,to obtain 2-benzoyl- 2-azabicyclo [2 .2.2] octan-S-one.

14 EXAMPLE 8 Oxygenation of 3- (p-toluenesulfonyl) -3 -azabicyclo[3.2.2]nonane The biconversion and extraction procedures of Example 1are repeated using 2 g. of 3-(p-toluenesulfonyl)-3-azabicyclo[3.2.2]nonane as the substrate.

The extract residue thus obtained is chromatographed on a column ofFlorisil. The column is eluted with Skellysolve B containing increasingproportions of acetone and those fractions which are shown to containthe desired products by thin layer chromatography are combined andevaporated to dryness to give 3-(p-toluenesulfonyl)-3-azabicyclo[3.2.2]nonan-6-one and 3-(p-toluenesulfonyl)- 3 -azabicyclo [3.2.2]nonan-6-ol.

The 3-(p-toluenesulfonyl) 3-azabicyclo[3.2.2]nonan- 6-01 thus obtainedis oxidized with Jones reagent in accordance with the proceduredisclosed in Example 2, above, to obtain 3 (p-toluenesulfonyl)3-azabicyclo [3.2.2]nonan-6-one.

EXAMPLE 9 Oxygenation of bispentamethyleneurea The biconversion andextraction procedures of Example 1 were repeated using 5 g. ofbispentamethyleneurea as the substrate. The extract residue thusobtained was chromatographed on Florisil. Elution with Skellysolve Bcontaining increasing proportions of acetone and acetone gavel-pentamethylenecarbamyl 4-hydroxypiperidine in the 25acetone-Skellysolve B eluates and early acetone eluates. These fractionswere combined, evaporated to remove the solvent, redissolved in acetoneand oxidized with excess Jones chromic acid reagent. The keto materialwas chromatographed on Florisil. Elution with 10% acetone-Skellysolve Band later 25% acetone-Skellysolve B aflYorded the desiredl-pentamethylenecarbamyl 4 oxopiperidine, which was recrystallized fromSkellysolve B several times to M.P. 66-67" C.

Analysis.-Calcd. for C H N O (percent): C, 62,83; H, 8.63; N, 13.32.Found (percent): C, 62.84; H, 8.36; N, 13.35.

EXAMPLE 10 Oxygenation of 1-benzoy1-2-methyl-5-ethylpiperidine Thebioconversion and extraction procedures of Example l were repeated using2 g. of 1-benzoyl-2-methyl- 5-ethylpiperidine as the substrate. Theresidue thus obtained was chromatographed on a column of Florisil andthe column was eluted with Skellysolve B containing increasing portionsof acetone. The eluate fractions were analyzed by thin layerchromatography and those containing the desired product were combinedand the solvent removed by evaporation to give 1-benzoyl-2-methy1 4-hydroxy-S-ethylpiperidine.

The product thus obtained was oxidized with Jones reagent in the mannerdisclosed in Example 2, above, to give1-benzoyl-2-methyl-4-oxo-S-ethylpiperidine.

EXAMPLE 11 Oxygenation of 3-benzoyl-3-azabicyclo [3 .2.2]nonane A mediumwas prepared of 2.5 kg. of cornsteep liquor (60% solids), 1 kg. ofcommercial dextrose, and l. of tap water, and adjusted to a pH between4.8 and 5. This medium was sterilized, inocuated with a 72-hourvegetative growth of Sporotrichum sulfurescens, ATCC 7159, and incubatedfor 19 hours at a temperature of about 28 C. using a rate of aeration of6.25 l. per minute at about 300 rpm. The substrate, 25 g. of 3-benzoyl-3-azabicyclo[3.2.2]nonane in solution in a minimum amount of acetone(about 250 ml.), was then added to the fermentation and incubation Wascontinued for an additional 72-hour period under the same conditions.Phenolized lard oil (5 g. phenol per liter of lard oil) was added as anantitoam agent as needed, about 50 ml.

was used during the bioconversion. The beer and mycelium were thenseparated by filtration and the mycelium was washed with water and thewash-water was added to the beer filtrate. The thus obtained beerfiltrate was extracted four times with a volume of methylene chlorideequal to one-fourth the volume of the filtrate. The combined extractswere washed with one-fourth volume of deionized water and the solventwas removed by distilla tion at reduced pressure to give 25.8 g. ofresidue, which was analyzed by thin layer chromatography and gasliquid(vapor phase) chromatography and found to contain unconverted substrate,3-benzoyl-3-azabicyclo[3.2.2] nonane-6-one and 3-benzoyl-3-azabicyclo [3.2.2] nonane- 6-ol. Gas chromatography showed that the residue containedunchanged substrate, 6-ketone, and G-hydroxyl in the proportions of124332833.

The extract residue from the bioconversion (25.8 g.) was dissolved in 50ml. of acetone and oxidized by the addition of 32 ml. of Jones reagentover a 5 minute period keeping the temperature of the solution below 30C. After the addition, the mixture was allowed to stir for ten minuteslonger with no temperature control and excess oxidant was then destroyedby the addition of 1 ml. of isopropanol. After adding 1 l. of water andstirring to dissolve sludge, the mixture was extracted three times with250 ml. of methylene chloride each time. The combined extract was washedonce with 250 ml. of Water, dried over sodium sulfate, and the solventremoved by distillation, applying reduced pressure at the later stage.The yield of crude residue as an oil was 23.2 g. Gas chromatography andthin layer chromatography showed the oil to be mainly3-benzoyl-3-azabicyclo[3.2.2] nonan-6-one containing a small amount ofunconverted substrate. This material was dissolved in 250 ml. ofmethylene chloride and percolated through a bed of 175 g. of Florisil,eluting with 500 ml. of methylene chloride followed by 3000 ml. ofSkellysolve B containing 18% acetone. The combined eluate was taken todryness and the-residual oil was distilled through a 4 inch Vigreuxcolumn at reduced pressure to give 12.17 g. of S-benzoyl-3-azabicyclo[3.2.2]nonan-6-one, B.P. 190-195 0.3 torr.

The following derivatives of 3-benzoyl-3-azabicyclo-[3.2.2]-nonane-6-one were prepared to confirm the structure.

Semicarbazone: M.P. l97200 C.

Analysis.Calcd for C H N O (percent): C, 63.98; H, 6.71; N, 19.65. Found(percent): C, 64.06; H, 6.82; N, 18.29.

Oxime: M.P. 156-158 C.

Analysis.Calcd for C H N O (percent): C, 69.74; H, 7.02; N, 10.85. Found(percent): C, 69.71; H, 7.16; N, 10.87.

2,4-dinitrophenylhydrazone2 M.P. 198-201" C.

Analysis-Calcd for C H N O (percent): C, 59.56; H, 5.00; N, 16.54. Found(percent): C, 59.57; H, 4.94; N, 16.65.

EXAMPLE 12 Oxygenation of benzyl-3-azabicyclo[3.2.2]nonane-3-carboxylate The bioconversion and extraction procedures of Example 11were repeated using 50 g. of benzyl-3-azabicyclo[32.2]nonane-3-carboxylate as the substrate. The beer extract residuethus obtained, containing benzyl-6-hydroxy 3 azabicyclo[3.2.2]nonane 3carboxylate, weighed 22.0 g. This was dissolved in 500 ml. of acetoneand 50 ml. of Jones reagent was slowly added while stirring andmaintaining the temperature between 25-30 C. during five minutes. Themixture was stirred ten minutes longer at about 28 C. and excess oxidantwas destroyed by the addition of 15 m1. of isopropanol. Most of theacetone was removed under reduced pressure, the mixture was diluted with500 ml. of water and extracted once with 200 ml. and four times with 100ml. volumes of methylene chloride. The combined extract was washed oncewith 250 ml. of water, dried over sodium sulfate, and the solventremoved to give 19.1 g. of oil, which was chromatographed over 500 g. ofFlorisil, placing it on the column with 150 ml. of methylene chlorideand eluting by the gradient method with 8.1 of Skellysolve B plusincreasing proportions of acetone from 0 to 20% and collecting fractionsof about 375 ml. each. The fractions were examined by infrared.Fractions 7-11 contained unchanged substrate. Fractions 14-20 containeda ketone conversion product identified as one component by vaporphasechromatography. Fractions 14-20 containing the desired product werecombined and the solvent removed by distillation at reduced pressure togive 8.93 g. of benzyl- 6-oxo-3-azabicyclo[3.2.21-nonane 3 carboxylateas a yellow oil.

AnaZysis.Calcd. for C H NO (percent): C, 70.31; H, 7.01; N, 5.13. Found(percent): C, 70.58; H, 7.41; N, 5.53.

The 2,4-dinitrophenylhydrazone derivative of thebenzyl-6-oxo-3azabicyclo[3.2.2]nonane-3 carboxylate melted at 84-86 C.

AnaZysis.--Ca1cd. for C H N O (percent): C, 58.27; H, 5.11; N, 15.45.Found (percent): C, 58.43; H, 5.21; N, 15.32.

EXAMPLE l3 Oxygenation of l-benzoyloctamethyleneimine The bioconversionand extraction procedures of EX- ample 11 were repeated using 15 g. ofl-benzoyloctamethyleneimine as the substrate. The extract residue thusobtained, containing mainly 1-benzoyl-4-hydroxyoctamethyleneimine and1-benzoyl-5-hydroxy-octamethyleneimine, was taken up in acetone (ca. 1liter) and oxidized with excess Jones reagent. The resultant ketoamidemixture was chromatographed on Florisil. The less polar ketoamide,4-oxo-l-benzoyloctamethyleneimine, was eluted with 25%acetone-Skellysolve B, and the more polar ketoamide,5-oxo-1-benzoyloctamethyleneimine, was eluted with acetone.

EXAMPLE 14 Oxygenation of 1-benzoyloctamethyleneimine The bioconversionand extraction procedures of EX- ample 11 were repeated using 25 g. ofl-benzoyloctamethyleneimine as the substrate. The extract residue thusobtained was dissolved in 500 ml. of acetone. The solution was cooled onan ice bath, Excess l ones reagent was added dropwise to the solution.After 10 minutes, the excess Jones reagent was decomposed by theaddition of isopropyl alcohol. The mixture was concentrated to an oilyresidue, which was dissolved in 200 ml. of water and 100 ml. ofmethylene chloride. The aqueous phase was separated and extracted withmore methylene chloride (2X 100 ml.). The combined extracts were driedand concentrated. The residual material thus obtained was dissolved in100 m1. of methylene chloride and the solution was placed on a 2.5 kg.column of Florisil packed in Skellysolve B hexanes. The following twoliter fractions were collected: 2 of Skellysolve B, 10 of 10% (v./v.)acetone in Skellysolve B, 8 of 20% acetone in Skellysolve B, 5 of 50%acetone-Skellysolve B, and 3 of acetone. Fractions 15 through 18 gave4.80 g. of l-benzoyl-4-oxooctamethyleneimine as a crystalline solid andfractions 20 through 23 gave 11.62 g. of 1-benzoyl-5-oxooctamethyleneimine as a crystallne solid. A sample 1.60 g.) of the 1-benzoyl-4-oxooctamethyleneirnine was dissolved in methylene chloride andplaced on a column of Florisil g.) packed with Skellysolve B. Elutionwith 10% (v./v.) acetone in Skellysolve B gave colorless crystals. Tworecrystallizations from cold methylene chloride-Skellysolve B gave1-benzoyl-4-oxooctamethyleneimine as colorless plates, M.P. 87-88" C.;11 1700 m.s., 1625 s., 11, 787 m.s., 744 m.s., 705 m.s. cm.- in Nujol.

Analysis.-Calcd. for C H NO (245.31) (percent): C, 73.44; H, 7.81; N,5.71. Found (percent): C, 73.30; H, 8.03; N, 5.80.

A sample (11.40 g.) of the l-benzoyl-S-oxooctamethyleneimine dissolvedin methylene chloride was placed on a column of Florisil (600 g.) packedwith Skellysolve B. Elution with 25% (v./v.) acetone in Skellysolve Bgave colorless crystals, which were recrystallized fromacetone-Skellysolve B to M.P. 69-71 C. A final recrystallization fromacetone-Skellysolve B gave 1-benzoy1-5-oxooctamethyleneimine ascolorless crystals M.P. 70-72 C.; v 1700 m.s., 1625 s., 11,800 m., 748m.s., 717 s. cm.- in Nujol.

Analysis.Calcd. for C H NO (percent): C, 73.44; H, 7.81; N, 5.71. Found(percent): C, 73.54; H, 7.72; N, 5.94.

EXAMPLE 15 Oxygenation of 2-benzoyl-2-azabicyclo [2.2.2]octane Thebioconversion and extraction procedures of Example 11 were repeatedusing 25 g. of 2-benzoyl-2-azabicyclo [2.2.2]octane as the substrate.The extract residue thus obtained was dissolved in methylene chlorideand chromatographed over 1000 g. of Florisil. Elution was with 4 1. eachof Skellysolve B hexanes containing acetone and 12 l. of Skellysolve Bcontaining acetone; cuts of 800 ml. each were collected; pooled residuesas follows:

(A) Fractions 7-11=3.98 g. unchanged substrate (B) Fractions 17-18=1.61g. of 2-benzoyl-2-azabicyclo [2.2.2]octan-exo-6-ol (C) Fraction 19:2.14g. of mixture (D) Fractions 20-27=12.16 g. of2-benzoyl-2-azabicyclo[2.2.2]octan-exo-5-ol.

(B) above was recrystallized from acetone; yield 1.61 g. of2-benzoyl-2-azabicyclo[2.2.2]octan-exd6-ol, M.P. 200-205 C.

Analysis.--Calcd. for C H NO (percent): C, 72.70; H, 7.41; N, 6.03.Found (percent): C, 72.70; H, 7.64; N, 5.82.

(D) above was recrystallized from acetone; yield, 10.62 g. of2-benzoyl-2-azabicyclo[2.2.2]octan-exo-5-ol, M.P. 146-148 C.

Analysis.-Calcd. for C H NO (percent): C, 72.07; H, 7.41; N, 6.03. Found(percent): C, 72.52; H, 7.19; N, 6.03.

EXAMPLE l6 Oxygenation of 3-benzoyl3-azabicyclo [3 .3 1 nonane Thebioconversion and extraction procedures of Example 11 were repeatedusing 25 g. of 3-benzoyl-3-azabicyclo[3.3.1]nonane as the substrate. Theextract residue thus obtained was chromatographed on Florisil (2.0 kg.)packed with Skellysolve B. Elution with 25 and (v./v.)acetone-Skellysolve B gave crystalline material (19.645 g.).Recrystallization from acetone proceeded by decolorization withactivated charcoal gave colorless crystals, M.P. 139-141 C. From theseveral crops collected, a total of 16.894 g. (0.0689 mole, 63%) of3-benzoyl-3- azabicyclo[3.3.1]n0nan-axial-6-ol was obtained, M.P.137-141 C. Three recrystallizations from acetone gave 3 benzoyl 3azabicyclo[3.3.1]nonan-axial-6-ol, M.P. 139l41 C.; [0:1 1 (0., 0.860,chloroform); 11 3460, 3420 sh., v g 02C 1610, 1575, 1525, 1495, 11,785,740, 705 cm.- in Nujol.

Analysis.-Calcd. for C H NO (percent): C, 73.44; H, 7.81; N, 5.71. Found(percent): C, 73.66; H, 7.96; N, 6.07.

3-benzoyl-3-azabicyclo[3.3.1]n0nane was bioconverted in accordance withthe procedure described above, using the microorganism Rhizopusarrhizis, ATCC 11145 (American type culture collection), to give3-benzoyl-3- azabicyclo[3.3.1]nonan-1-ol, M.P. 138-140 C.; [a1

40 (c., 0.803, chloroform); v 3330, 11 C=c 1600, 1575, 1520, 1495, v,725, 705 cm.- in Nujol.

Analysis.Ca1cd. for C H NO (percent): C, 73.44; H, 7.81; N, 5.71. Found(percent): C, 73.46; H, 7.76; N, 6.15, and3benzoyl-axial-6-hydroxy-3-azabicyclo[3.3.1] nonane, M.P. -137 C.,having an infrared spectrum identical with that of the hydroxylatedproduct isolated from bioconversion with Sporotrichum sulfurescens,above.

EXAMPLE 17 Oxygenation of 1-benzoyl-2-propylpiperidine The bioconversionand extraction procedures of Example 11 were repeated using 25 g. of1-benzoyl-2-propylpiperidine as the substrate. The extract residue thusobtained was dissolved in methylene chloride and chromatographed onFlorisil to give 0.524 g. of 1-benzoyl-4- 'hydroxy-Z-propylpiperidinewhich was recrystallized from acetone-hexanes to M.P. 124-130 C. (0.382g.)

Analysis.-Ca1cd. for C H NO (percent): C, 72.84; H, 8.56; N, 5.66. Found(percent): C, 72.64; H, 8.71; N, 5.44, and 0.674 g. of1-benzoyl-2-(2-hydroxypropyl)piperidine which was recrystallized fromacetone-hexanes to M.P. 1l6119 C.

Analysis.Ca1cd. for C H NO (percent): C, 72.84; H, 8.56; N, 5.66. Found(percent): C, 73.09; H, 8.47; N, 5.59.

A 0.157 g. sample of the 1-benzoyl-4-hydroxy-2-propylpiperidine wasdissolved in acetone and oxidized with excess Jones reagent to give1-benzoyl-4-oxo-2-propylpiperidine.

A 0.160 g. sample of the 1-benzoyl-2-(Z-hydroxypropyl)piperidine wasdissolved in acetone and oxidized with excess Jones reagent to give1-benzoyl-2-(2-oxopropyl)piperidine.

EXAMPLE 18 Oxygenation of 1-benzoyl-4-propylpiperidine The bioconversionand extraction procedures of Example 11 were repeated using 25 g. of1-benzoyl-4-propylpiperidine as the substrate. The extract residue thusobtained was chromatographed on Florisil and eluted with hexanescontaining increasing proportions of acetone. The appropriate fractionsdetermined by NMR and IR were combined and distilled to give1-benzoyl-4-(2-oxopropyl) piperidine, B.P. 168-172 C. (0.15 mm.); N1.5441.

EXAMPLE 19 Oxygenation of 1-benzoyl-2-methylpiperidine The bioconversionand extraction procedures of Example 11 were repeated using 25 g. of1-benzoyl-2-methylpiperidine as the substrate. The extract residue thusobtained was chromatographed on Florisil. The fractions containing the4-oxo product were combined and recrystallized from acetone-hexanes togive 0.252 g. which was recrystallized again from acetone-hexanes togive l-benzoyl-4-oxo-2-methylpiperidine, M.P. 117118 C.

Analysis.-Calcd. for C H NO (percent): C, 71.86; g1, 6.96; N, 6.45.Found (percent): C, 72.21; H, 7.07; N,

The fractions from the chromatogram which contained the 3-hydroxyproduct were combined and recrystallized from acetone-hexanes to give1.225 g. of product which.

was twice recrystallized from acetone-hexanes to give 1-benzoyl-3-hydroxy-2-methylpiperidine, M.P. 127-129 Analysis.Calcd. forC13H17NO2 (percent): C, 71.20; H, 7.82; N, 6.39. Found (percent): C,71.26; H, 8.00; N, 6.68.

The fractions containing the 4-hydroxy product were combined andrecrystallized from acetone-hexanes to give 4.277 g. of product whichwas recrystallized from acetonehexanes to give1-benzoyl-4-hydroxy-2-methylpiperidine, M.P. l24125 C.; [a] 29.

Analysis.--Calcd. for C H NO (percent): C, 71.20; H, 7.82; N, 6.39.Found (percent): C, 71.25; H, 7.83; N, 6.65.

A 0.352 g. sample of the 1-benzoyl-3-hydroxy-2-methylpiperidine in ml.of acetone was oxidized with excess Jones reagent and concentrated on asteam bath. Water was added and the solution was extracted wtihchloroform (3 X 15 ml.). The combined extracts were dried of magnesiumsulfate and concentrated under reduced pressure to an oil which wascrystallized from methylene chloride-hexanes to give 0.163 g. of1-benzoyl-3-oXo-2- methylpiperidine, M.P. 102104 C., which wasrecrystallized to give colorless plates, M.P. 103-105 C.; [od +74.

Analysis.-Ca1cd. for -C H N0 (percent): C, 17.86; H, 6.96; N, 6.45.Found (percent): C, 71.99; H, 7.09; N, 6.25. I

A 0.5 g. sample of 1-benzoyl-4 hydroxy-2-methylpiperidine was dissolvedin 25 ml. of acetone, oxidized and worked up in the manner describedabove to give 0.325 g. of 1-benzoyl-4-oxo-2-methylpiperidine, M.P.116-119? C. The infrared spectrum was identical to the same materialobtained directly from the bioconversion, above.-

EXAMPLE Oxygenation of 1-benzoyl-4-methylpiperidine The bioconversionand extraction procedures of Example 11 were repeated using g. of1-benzoyl-4-methylpiperidine as the substrate. The extract residue thusobtained was chromatographed on Florisil. The fractions containing the4-hydroxy product were combined and recrystallized from acetone-hexanesto give 1.342 g. which was recrystallized twice again fromacetone-hexanes to give 1-benzoyl-4-hydroxy4-methylpiperidine, M.P. 104-106 C.

Analysis.'Calcd. for C H NO (percent): C, 71.20; H, 7.82; N, 6.39. Found(percent): C, 71.24; H, 7.77; N, 6.53.

The fractions from the chromatogram which contained the hydroxy methylproduct were combined and recrys stallized from acetone-hexanes to give4.721 g. of product which was twice recrystallized from acetone-hexanesto give 1 -benzoyl 4 hydroxymethylpiperidine, M.P. 92- 95 C.

Analysis.Calcd. for C H NO (percent): C, 71.20; H, 7.82; N, 6.39. Found(percent): C, 71.37; H, 7.94; N, 6.43.

EXAMPLE 21 3-benzoyl-3-azabicyclo 3 .3 1]nonan-6-one A solution of3-benzoyl-3-azabicyclo[3.3.1]nonan-axial-6-ol (1.543 g., 0.00630 mole)in acetone (150 ml.) was cooled on an ice bath and treated with anexcess (1.8 ml.) of Jones reagent. After minutes at room temperature,the excess oxidant was consumed with isopropyl alcohol. The solution wasdecanted and filtered through sodium sulfate. The residue was dissolvedin water and extracted with methylene chloride. The combined organicsolutions were dried over magnesium sulfate, Celite was added, and themixture again filtered. The filtrate was concentrated under reducedpressure and cooled. Crystallization gave 0.882 g. (0.00363 mole, 57%)of product, M.P. 158-160 C. Two recrystallizations from acetone gavecolorless crystals of 3-benzoyl-3-azabicyclo [3.3.1]nonan-6-one, M.P.159-l61 C.; v 1705, 1620, v 1600, 1580, 1570, 1490, v, 785, 775, 735,700 cm."- in Nujol.

Analysis.-Calcd. for C H NO (percent): C, 74.05; H, 7.04; N, 5.76. Found(percent): C, 73.16; H, 7.30; N, 5.29.

EXAMPLE 22 2-benzoyl-2-azabicyclo[ 2.2.2] octan-S-one Two grams of2-benzoyl-2-azabicyclo[2.2.2]octan-5-ol dissolved in 100 ml. of acetonewas treated dropwise with a slight excess of a stock solution ofoxidizing reagent prepared from 26.7 g. of chromium trioxide, 50 ml. ofwater, and 23 ml. of concentrated sulfuric acid (Jones reagent). An icebath was applied as necessary to keep the temperature below 30 duringthe oxidation. Isopropanol (5.0 ml.) was added to destroy excess chromicacid. The mixture was diluted with 200 ml. of water and extracted withmethylene chloride. The extract was Washed once with water and driedover sodium sulfate. The solvent was removed by distillation andresulting product crystallized to yield 1.95 g. ofZ-benzoyl-Z-azabicyclo[2.2.2]octan-5-one, M.P. 6672 C.; IR (CHClsolution), 1740 cm.- (C=O).

@EXAMPLE 23 Z-benzoyl-Z-azabicyclo[2.2.2] octan-6-one A mixture of 300mg. of 2-benzoyl-2-azabicyclo[2.2.2] octan-6-ol (300 mg.) in 20 ml. ofacetone was oxidized with chromic acid in the manner described in'Example 22, above. The product thus obtained was recrystallized fromacetone-hexane to give 2-benzoyl-2-azabicyclo [2.2.2]octan-6-one, M.P.99101 C.; IR (CHCl solution), 1740 cm.- (0:0).

EXAMPLE 24 1-benzoyl-5-hydroxyheptamethyleneimine A solution of 700 mg.of 1-benzoyl-5-oxoheptamethyleneimine (from Example 5, above) in 12 ml.of methanol was reduced with a solution of mg. of sodium borohydride in2 ml. of N/ 10 sodium hydroxide at room temperature overnight. Theproduct was recovered by acidification with acetic acid, evaporation ofmost of the methanol at reduced pressure, and partition between waterand methylene chloride. Chromatography of the organic solution onFlorisil gave 657 mg. of l-benzoyl-S-hydroxyheptamethyleneimine in theacetone eluate; for analysis the material was recrystallized fromacetone-Skellysolve B to M.P.116-118 C.

Analysis.Calcd. for C H NO (percent): C, 72.07; H, 8.21; N, 6.00. Found(percent): C, 72.18; H, 8.01; N, 6.03.

In the same manner following the procedure of Example 23, other ketocompounds of this invention can likewise be reduced to the correspondinghydroxy compounds by substituting the appropriate keto compound asstarting material in place of l-benzoyl-S-oxoheptamethyleneimine. Thefollowing conversions are representative:

1-benzoyl-4-oxoheptamethyleneimine to 1-benzoyl-4-hydroxyheptamethyleneimine;

1 (p-toluenesulfonyl)-4-oxohexamethyleneimine to 1- p-toluenesulfonyl)-4-hydroxyhexamethyleneimine;

1 benzoyl-5-oxododecamethyleneimine to l-benzoyl-S-hydroxydodecamethyleneimine;

1 benzoyl-6-oxododecamethyleneimine to 1-benzoyl-6-hydroxydodecamethyleneimine;

1 .benzoyl-7-oxododecamethyleneimine to 1-benzoyl-7-hydroxydodecamethyleneimine;

1 benzoyl 4-oxooctamethyleneimine to 1-benzoyl-4-hydroxyoctamethyleneimine;

1 benzoyl 5-oxooctamethyleneimine to l-benzoyl-S-hydroxyoctamethyleneimine;

3 (p toluenesulfonyl) 3-azabicyclo[3.2.2]nonan-6- one to3-(p-toluenesulfonyl)-3-azabicyclo[3.2.2]nonan-6- ol; and the like.

EXAMPLE 25 3*benzoy1-3-azabicyclo-[3.3.1]nonan-equatorial-6-ol Asolution of sodium borohydride (1.0 g., 0.0265 mole) in 10 ml. of 0.1 Maqueous sodium hydroxide was added to a solution of3-.benzoyl-3-azabicyclo[3.3.1]nonan-6-one (1.017 g., 4.18 mmoles) in 40ml. of methanol. Thin layer chromatography after 0.5 hour showed thereaction to be complete. The solution was partially concentrated underreduced pressure and then was diluted with 150 ml. with water. Thesolution was made acidic (pH -6) with acetic acid and was concentratedunder reduced pressure over a hot water bath until crystals began toform. The mixture [was extracted with methylene chloride (3 X 50 ml.).From the dried (magnesium sulfate) extract solution, an oil was obtainedfollowing concentration. The oil crystallized and the solid wasrecrystallized from acetone-Skellysolve B, giving 0.695 g. (2.84 mmoles,67%) of 3-benzoy1- 3-azabicyclo[3.3.ljnonan-equatorial-6-ol, M.P.135-138 C., which was recrystallized twice from acetone-skellysolve B toM.P. 139-141 C.; VOH 3360, v 1600, v 1590, 1575, 1530, 1490, 11 1060, a,790, 780, 735 705 cm.- in Nujol.

Analysis.Calcd. for C H NO (percent): C, 73.44; *H, 7.81; N, 5.71. Found(percent): C, 73.92; H, 7.95; N, 6.08.

EXAMPLE 26 3 benzoyl-3 -azabicyclo [3 .2.2] nonan-exo-6-ol Twenty gramsof the crude 3-benzoyl-3-azabicyclo[3.2.2]nonan-exo-6-one dissolved in350 ml. of methanol was treated with a solution of 16.0 g. of sodiumborohydride in 100 ml. of N/ 10 sodium hydroxide for 30 minutes. Thinlayer chromatography indicated complete reaction. The mixture wasdiluted with 300 ml. of water and allowed to stand for 18 hours withchilling. The mixture was then adjusted to pH 6 by the cautious additionof 50% acetic acid. The solid product thus obtained was recovered byfiltration, washed with water and dried; yield, 12.75 g. of3-benzoyl-3-azabicyclo[3.2.2]nonanexo-6-ol, M.P. 131-135" C.; ananalytical sample from acetone melted at 135137 C.

Analysis.Calcd. for C H NO (percent): C, 73.44; H, 7.81; N, 5.71. Found(percent): C, 73.31; H, 7.87; N, 5.89.

EXAMPLE 27 3-azabicyclo 3 .2.2] nonan-exo-6-ol 6-benzoate hydrochloride(acyl migration) To a warm solution of 3-benzoy1-3-azabicyclo[3.2.2]nonan-exo-G-ol (2.14 g.) in 50 ml. of tetrahydrofuran was added 4.0 ml.of concentrated hydrochloric acid and the mixture was allowed to standat C. The solution was examined at intervals by thin layerchromatography and after complete reaction (23 hours), it wasconcentrated under reduced pressure. The oil thus obtained was tr1-turated twice with ether, decanting off the ether each time, and the HClsalt was precipitated by adding 25 ml. of acetone and ml. of ether;yield, 2.16 g., M.P. 205-208 C. This was recrystallized frommethanol-methyl ethyl ketone; yield 2.06 g. of3-azabicyclo[3.2.2]nonan-exo-6-ol 6-benzoate hydrochloride, M.P. 205-208C.; IR amine, HCl at 2250-2700 cm.- ester C=O at 1710 cmrAnalysis.Calod. for C15H2QNO2C1 (-pl"Cnt)l C, 63.93; H, 7.15; N, 4.97.Found (percent) C, 63.92; H, 7.44; N, 5.24.

EXAMPLE 28 3-benzoyl-3 -azabicyclo 3.3 .1 .1 nonan-fi-one ethylene ketalA mixture of 3-benzoyl-3-azabicyclo[3.3.1]nonan-6-one (0.550 g., 0.00226mole) in benzene (100 ml.), p-toluenesulfonic acid hydrate (0.090 g.,0.000473 mole), and ethylene glycol (10 ml.) was heated to reflux for 18hours. The condensate was dried by passing through a calcium carbidetrap. A few drops of pyridine were added and the mixture was cooled toroom temperature. The mixture was extracted with 5% aqueous sodiumbicarbonate solution (50 ml.) and with water (2X25 ml.). The benzenelayer was dried over magnesium sulfate and concentrated under reducedpressure to give as 3-benzoyl-3-azabicyclo[3.3.1]- nonan-6-one ethyleneketal as an oil, 11 1630, 1162c 1605, 1580, 1500, v, 708 cm.-

22 EXAMPLE 29 8-benzoyl-1,4-dioxa-8-azaspiro[4.7]dodecane A mixture ofl-benzoyl-4-oxoheptametheneimine (16.487 g., 0.0712 mole),p-toluenesulfonic acid hydrate (1.35 g., 0.00710 mole), ethylene glycol(25 ml.), and benzene (200 ml.) was heated to reflux for 24 hours. Thecondensate was passed through a calcium carbide drying trap during thistime. Pyridine (2.0 ml.) was added and the mixture was cooled. Themixture was extracted with aqueous sodium bicarbonate solution and withwater (3 100 ml.). The benzene layer was dried over magnes'ium sulfateand concentrated to give 8-benzoyl-1,4- dioxa-S-azasporo[4.7]dodecane asa viscous oil.

EXAMPLE 3 O 9-benzoyl-1,4-dioxa-9azaspiro[4.71dodecane A mixture of1-benzoy1-S-heptamethyleneimine (39.883 g., 0.172 mole),p-toluenesulfonic acid (2.96 g., 0.0155 mole), ethylene glycol ml., 83g., 1.34 mole), and benzene (500 ml.) was heated to reflux. Thecondensate was dried by passing it through a calcium carbide trap. Themixture was refluxed 30 hours. Pyridine (6.0 ml.) was then added to thecooled mixture, the benzene layer was extracted with 5% aqueous sodiumbicarbonate (3 X 100 ml.) and dried over magnesium sulfate.Concentration of the benzene solution gave 42.95 g. (0.156 mole, yield)of a viscous oil which was crystallized from ether to give9-benzoy1-1,4-dioxa-9-azaspiro[4.7]dodecane, M.P. 72-74 C. Tworecrystallizations from ether- Skellysolve B gave colorless crystals of9-benzoyl-1,4- dioxa-9-azaspiro[4.7]dodecane, M.P. 72-73 C.; 11 1630 s.cm. in Nujol.

Analysis.-Calcd. for C H NO (275.34) (percent): C, 69.79; H, 7.69; N,5.09. Found (percent): C, 69.99; H, 7.84; N, 5.20.

EXAMPLE 31 9-benzoyl-1,4-dioxa-9-azaspiro [4.8]tridecane A solution of 1benzoyl-5-oxooctamethyleneimine (18.819 g., 0.0768 mole) andp-toluenesulfonic acid hydrate (1.46 g.) in benzene (200 ml.) withethylene glycol (30 ml.) was heated to the reflux temperature of benzenefor 22 hours and the condensate was passed through a calcium carbidedrying trap. Pyridine (3.0 ml.) was added at the end of the refluxperiod, the mixture was cooled and then extracted with 5% aqueous sodiumbicarbonate ml.) and with water (2x 100 ml.). The benzene layer wasdried over magnesium sulfate, and concentrated under reduced pressure,giving a viscous yellow oil. The infrared spectrum of the oil showed thepresence of a carbonyl function (1730 w. cmf and so the oil wasresubjected twice to the ketalization process. The crude oily productcrystallized spontaneously on standing. Recrystallization fromether-Skellysolve B gave two crops (13.682 g., 0.0473 mole, 61%) oflight yellow crystals, M.P. 9 0-95 C., which were recrystallized fromether- Skellysolve B to give 9-benzoyl-1,4-dioxa-9-azaspiro[4.8]tridecane, M.P. 98-100 C. A final recrystallization preceded bydecolorization with activated charcoal gave colorless needles of9-benzoyl-1,4-dioxa-9 azaspiro[4.8]

tridecane, M.P. 99'l0l C.; v 1630 5.; :1, 798 m., 737 m., 704 ms. cm.-in Nujol.

AnaZysis.Calcd. for C H NO (289.36) (percent): C, 70.56; H, 8.01; N,4.84. Found (percent): C, 70.50; H, 8.08; N, 5.16.

EXAMPLE 32 8-benzoyl-1,4-dioxa-8-azaspiro [4.6]undecane A mixture of11.957 g. (0.0551 mole) 1-benzoyl-4- oxohexamethyleneimine in 200 ml. ofbenzene, 1.020 g. (0.00537 mole) of p-toluenesulfonic acid hydrate and28 ml. of ethylene glycol was stirred and heated at reflux for about 24hours. The condensate was passed through a calcium carbide trap toremove water. Pyridine (3 ml.)

was added at the end of the reflux period and the mixture cooled. Themixture was extracted with 5% aqueous benzene layer was dried andconcentrated under reduced pressure to give 8benzoyl-l,4-dioxa-8-azaspiro[4.61undecane as an oil, 1 1630 cmr EXAMPLE33 S-benzyl-1,4-dioxa-8-azaspiro [4.6] undecane The product from Example32 above, was dissolved in 75 ml. of ether and added dropwise to amixture of lithium aluminum hydride (2.0 g.) and ether (200' ml.). Themixture was stirred and heated to reflux for 3 hours, stirred at roomtemperature for about 72 hours, and again heated to reflux for 2 hoursbefore the excess lithium aluminum hydride was decomposed by theaddition of ethyl acetate and water. The solids thus obtained wereremoved by filtration and were washed with ether. The filtrate andwashes were combined, dried and concentrated to an oil which wasdistilled to give 9.696 g. (0.0392 mole), 71% of8-benzyl-1,4-dioxa-8-azaspiro[4.6]undecane as a pale yellow oil, B.P.120-121 C. (0.13 mm.); m 1.5312; v 1600, 1580, 1490, v, 730,695 cmrAnalysis.Calcd. for C H NO (247.33) (percent): C, 72.84; H, 8.56; N,5.66. Found (percent): C, 73.22; H, 8.98; N, 6.11.

EXAMPLE 34 9-benzyl-1,4-dioxa-9-azaspiro [4.7]dodecane A solution of9-benzoyl-1,4-dioxa-9-azaspiro[4.7]dodecane (40.9 g., 0.148 mole) inether (3 00* ml.) was added slowly to a stirred mixture of lithiumaluminum hydride (6.0 g., 0.158 mole) and ether (200 ml.). The resultingmixture was stirred at room temperature for 16 hours and at refluxtemperature for 5 hours. The excess lithium aluminum hydride wasdecomposed with 1:1 acetonewater and with water. The inorganic solidswere collected on a filter and washed three times with ether. Thecombined ether solution was dried and concentrated to an oil (38.26 g.).Simple distillation gave 9-benzyl-l,4-dioxa- 9-azaspiro[4.7]dodecane asa colorless oil (33.72 g., 0.129 mole, 87% yield), B.P. 125127 C. (0.3mm.); 11, 1115 s. cm.-

Analysis.Calcd. for C H NO (261.35) (percent): C, 73.53; H, 8.87; N,5.36. Found (percent): C, 73.39; H, 8.31; N, 5.34.

EXAMPLE 35 S-benzyl-1,4-dioxa-8-azaspiro [4.7] dodecane TheS-benzoyl-1,4-dioxa-8-azaspiro[4.7]dodecane from Example 30, above, wasdissolved in ether (2 l.) and slowly added to a stirred mixture oflithium aluminum hydride (3.0 g., 0.0790 mole) and ether (200 ml.). Theexcess ether was allowed to boil 01?. The remaining oil was dissolved intetrahydrofuran and added to the reaction mixture. The mixture wasstirred and refluxed 7 hours and then kept at room temperatureovernight. The excess lithium aluminum hydride was destroyed by additionof ethyl acetate and water. The inorganic solids were removed byfiltration and were washed with ether. The combined ether filtrates werecombined, dried, and concentrated to a yellow oil. Distillation of theoil gave 8 benzyl-1,4-dioxa-8-azaspiro[4.7]dodecane (5.700 g., 0.0218mole, 30% yield) as colorless oil, B.P. 140150 C. (0.2 mm.); 11 1.5284;11 1600 m.w., 1580 w., 1490 s., v, 725 s., 695 s. cmf

Analysis.Calcd. for C H NO (261.35) (percent): C, 73.53; H, 8.87;N,5.36. Found (percent): C, 73.33; H, 8.91; N, 5.14.

EXAMPLE 36 9-benzyl-1,4-dioxa-9azaspiro[4.8] tridecane A solution of 9benzoyl-1,4-dioxa-9-azaspiro[4.8] tridecane (13.331 g., 0.0462 mole) inether (250 ml.) was dribbled into a mixture of lithium aluminum hydride(2.0 g.) in ether ml.). The mixture was stirred at room temperature for16 hours and at reflux temperature for -4 hours. The excess hydride wasdecomposed by the cautious addition of water. The inorganic solids wereremoved by filtration and washed with ether. The combined ether solutionwas dried and concentrated to an oil. A simple distillation of the oilgave 9-benzyl-l,4- dioxa-9-azaspiro[4.8]tridecane (10.779 g., 0.0392.mole, 85% yield) as a colorless oil, B.P. 147 C. (1.5 mm.); n 1.5302; 111600 w., 1498 m., I, 750 m., 715 m.s., 701 ms. CHLTI. Analysis.-Calcd.for C17H25NO2 (275.38) (percent): C, 74.14; H, 9.15; N, 5.09. Found(percent): C, 74.77; H, 9.66; N, 5.04.

The product crystallized when kept overnight in the refrigerator, M.P.4446 C.

EXAMPLE 37 3-benzyl-3-azabicyclo[3.3.1]nonan-6-one ethylene ketal Asolution of the 3-benzoyl-3-azabicyclo[3.3.1]n0nan- 6-one ethylene ketalfrom Example 29, above, in ether was reduced with lithium aluminumhydride (0.5 g.) in ether. After refluxing 4 hours, the excess hydridewas decomposed with ethyl acetate and water, the solids were filteredoff, the ether solution was dried over magnesium sulfate andconcentrated to give 3-benzyl-3-azabicyclo [3.3.1]nonan-6-one ethyleneketal as an oil; 11 2930, 2900, 2875, 11 1600, 1580, 1495, v, 736, 700cmr EXAMPLE 38 3-benzyl-3-azabicyclo[3.3.1lnonan-l-ol A suspension of3-benzoyl-1-hydroxy-3-azabicyclo [3.3.1]n0nane (0.478 g., 0.00195 mole)in ether was added to a mixture of lithium aluminum hydride (0.6 g.) andether. The resulting mixture was stirred at room temperature overnightand then heated to reflux temperature for 7 hours. The excess hydridewas decomposed by addition of ethyl acetate and water. The solids wereremoved by filtration through celite and the ether solution was dried(magnesium sulfate). An oil was obtained upon removal of the ether. Theoil crystallized in the cold and crystals formed from a cold pentanesolution and were collected giving 0.206 g. (0.00892 mole, 45%) ofcrystals. M.P. 74-76 C. Recrystallization, preceded by decolorizationwith activated charcoal gave colorless crystals of3-benzyl-3-azabicyclo[3.3.1]nonan-1-ol, M.P. 73- 76 0.; [ed 24 (c.,0.740, CHCI 11 3280, 3210, v 1600, 1-585, 1495, 1 760, 745, 710, 700emfin Nujol; NMR confirmed the structure.

Analysis.-Calcd. for C H NO (percent): C, 77.88; H, 9.15; N, 6.05. Found(percent): C, 77.84; H, 9.45; N, 6.13.

EXAMPLE 39 3 -benzyl-3-azabicyclo[3 .2.2] nonan-endo-6-0l hydrochlorideThe 3 benzoyl 3 azabicyclo[3.2.2]nonan-endo-6-ol (6.69 g.) was dissolvedin 80 ml. of tetrahydrofuran and added with stirring to a mixture of 6.0g. of lithium aluminum hydride in 100 ml. of ether. The mixture wasrefluxed for one hour, chilled in a cold bath, and carefully decomposedby the addition of 25 ml. of water. After dilution with 300 ml. of etherand filtering, the filtrate and ether wash was dried (MgSO and thesolvent removed under reduced pressure to give 5.90 g. of 3-benzyl-3-azabicyc1o[3.2.2]nonan-endo-6-ol as a straw colored oil. Part oftheoil (1.33 g.) was dissolved in ether and treated with ethereal hydrogenchloride to precipitate the salt which was recrystallized from methanolether to give 1.07 g. of 3-benzyl-3-azabicyclo[3.2.2]nonan-endo-6-olhydrochloride, M.P. -l87 C.

Analysis.Calcd. for C H NOCl (percent): N, 5.23; Cl, 13.24. Found(percent): N, 5.47; Cl, 13.74.

25 EXAMPLE 4o 3-benzyl-3-azabicyclo[3.3.1]nonan-axial-6-ol A solution of3-benzoyl-3-azabicyclo[3.3.1]nonan-axial-6-ol (5.0 g., 0.204 mole) intetrahydrofuran (100 ml.) Was poured into a mixture of lithium aluminumhydride (3.0 g.) and tetrahydrofuran (150 ml.). The whole mixture washeated at reflux temperature for five hours then the excess hydride wasconsumed with ethyl acetate and water. The inorganic solids were removedby filtration and washed with hot tetrahydrofuran. The tetrahydrofuransolution was dried over magnesium sulfate and concentrated under reducedpressure to an oil. The oil was transferred with ether to a distillationflask. After a few minutes at low pressure, the oil solidified. Thesolid crystallized from cold hexane, giving crystals,'M.P. 67-69 C.Three recrystallizations from cold hexane, the last preceded bydecolorization with activated charcoal, resulted in colorless crystalsof 3-benzyl-3-azabicyclo[3.3.1]nonanaxial-6-ol, M.P. 70-71" C.; v 3320,3220, t 1600, 1495, v, 730, 695 cmr on the oil.

Analysis.-Calcd. for C H NO (percent): C, 77.88; H, 9.15; N, 6.05. Found(percent): C, 78.01; H, 9.54; N, 6.33.

EXAMPLE 41 3-benzyl-3-azabicyclo [3 .2.2] nonan-6-one hydrochloride The3-benzoyl-3-azabicyclo[3.2.2]nonan-6-one (5.13 g.), 60 ml. of benzene, 8ml. of pyrrolidine, and 100 mg. of p-toluenesulfonic acid was heated atreflux under a water trap for two hours. The mixture was concentratedunder reduced pressure to a dark oil. A solution of the crude enamine in50 ml. of ether, and 25 ml. of tetrahydrofuran was added to a stirredmixture of 3.0 g. of lithium aluminum hydride and 100 ml. of ether.After stirring at reflux for'one hour the mixture was chilled andcautiously treated with 15.0 ml. of water, diluted with ether, andfiltered. The-filtrate was dried (MgSO and concentrated to yield a lightyellow oil. Infrared indicated that the enamine protective moiety hasbeen replaced by carbonyl. The oil thus obtained was dissolved in etherand treated with ethereal HCl to precipitate 3-benzyl-3-azabicyclo[3.2.2]nonan-6-one hydrochloride.

Analysis.-Calcd. for C H NO-HCl (percent): N, 5.27; Cl. 13.34. Found(percent): N, 5.57; CI, 13.55.

EXAMPLE 42 3-benzyl-3-azabicyclo [3 .2.2] nonan-4-ol The crude3-benzy1-3-azabicyclo[3.2.2]nonan 4-ol resulting from the hydridereduction of 10 g. of 4-hydroxy- 3-benzoyl-3-azabicyclo[3.2.2]nonane,dissolved in 600 ml. of toluene and 150 ml. of cyclohexanone wasdistilled to remove ca. 100 ml. of toluene. Aluminum isopropoxide (20g.) was added, the mixture was distilled to remove ca. 50 ml. of solventand then heated at reflux for 60 minutes. After cooling it was pouredonto an ice mixture containing excess hydrochloric acid, stirred, andthe layers were separated. The aqueous acid layer was extracted severaltimes with ether and then made basic with 50% sodium hydroxide solution.The resulting emulsion was well extracted with ether and the etherextract was washed once with water and dried over magnesiumv sulfate.The ether solution was made up to 650 ml. and 100 ml. of this wastreated with ethereal HCl to precipitate the salt, which wasrecrystallized from methanol-ether to give 0.744 g. of3-benzy1-3-azabicyclo[3.2.2]nonan-6-one hydrochloride.

Analysis.Calcd. for C H NO-HCl (percent): C, 67.78; H, 7.59; N, 5.27;CI, 13.34. Found (percent): C, 68.13; H, 7.77; N, 5.42; Cl, 13.17.

EXAMPLE 43 3benzyl-3-azabicyclo 3 .3 .1 ]nonan-6-one A solution of3-benzyl-3-azabicyclo[3.3.1]nonan-6-ol obtained by lithium aluminumhydride reduction of 7.457

g. of 3-benzoyl-3-azabicyclo[3.3.1]nonan-6-one in toluene (120 ml.) andcyclohexane (30 ml.) was heated to boiling and the toluene-waterazeotrope distilled off. Aluminum i-propoxide (10 g.) and cyclohexanone(10 ml.) were added to the solution and the mixture was heated at refluxtemperature for two hours. The mixture was poured into ice-aqueoushydrochloric acid and stirred. The aqueous layer was separated,extracted with ether (3X ml.), and made alkaline with concentratedsodium hydroxide solution. A heavy precipitate formed at theneutralization point but disappeared and an oil formed as additionalbase was added. The solution and oily phase were extracted (3X 100 ml.)with ether; the ether was dried over magnesium sulfate and concentratedunder reduced pressure to a reddish-brown oil. The oil was transferredwith ether to a 10 ml. distillation flask and distilled, B.P. 126-429 C.(0.04 mm.), giving 4.171 g. (0.0182 mole, 60% yield) of3-benzyl-3-azabicyclo- [3.3.1]nonan-6-one of colorless oil, 11;, 1.5499;11 1700, 11 1600, 1580, 1490, 11,735, 695 cm. on the oil.

Analysis.Calcd. for C H NO (percent): C, 78.56; H, 8.35; N, 6.11. Found(percent): C, 78.90; H, 8.56; N, 6.05.

EXAMPLE 44 3-benzyl-3-azabicyclo[ 3 .3 .1 1 nonan-6-one perchlorate From3-ben2yl-3-azabicyclo 3 .3 l nonan-6-one ethylene ketal.-Aqueousperchloric acid (70%, 15 drops) was added to a solution of3-benzyl-3-azabicyclo[3.3.11- nonan-6-one ethylene ketal (0.25 g.,0.000915 mole) in absolute ethanol (5.0 ml.). The solution was heated onthe steam bath for three minutes. Addition of ether slowly precipitatedan oily solid, which crystallized into colorless crystals (0.251 g.,0.000763 mole, 83%), M.P. 210 215 C. Two recrystallizations from ethanolcontaining a few drops of water gave crystals of 3-benzyl-3-azabicyclo[3.3.1]nonan-6-one perchlorate, M.P. 2132l6 C.; VNH +3080, 11 1695, u1500, 11,, 770, 745, 705 cm.- in Nujol.

Analysis.-Calcd. for C H NO Cl (percent): C, 54.63; H, 6.11; N, 4.25.Found (percent): C, 54.51; H, 5.87; N, 4.33.

From 3-benzyl 3 azabicyclo[3.3.1]nonan-6-one.- Aqueous perchloric acid(70%, 10 drops) was added to a solution of 3-benzyl 3azabicyclo[3.3.1]nonan-6-one (0.236 g., 0.00103 mole) in absoluteethanol (3 ml.). Crystals formed after 10 minutes. A first crop of 0.157g. of colorless crystals was collected by filtration. A second crop of0.042 g. (0.199 g. total, 0.000605 mole, 58%) was obtained from themother liquor. The infrared spectrum of the crystals of 3-benzyl3-azabicyclo-[3.3.l]- nonan-6-one perchlorate thus obtained wasidentical to that of the above salt.

EXAMPLE 45 3-benzoyl-7-bromo-3-azabicyclo[ 3 .3 1 nonan- 6-one M.P. 141144" C. Two recrystallizations, the last preceded by decolorization withactivated charcoal, from acetone- Skellysolve B gave colorless crystalsof 3 benzoyl 7- bromo-3-azabicyclo[3.3.l]nonan-6 one, M.P. 147-149 C.;11 1715, 1630, v 1590, 1575, 1490, v, 730, 715, 700 cm? in Nujol.

27 Analysis.-Calcd. for C H BrNO (percent): C, 55.91; H, 5.01; N, 4.35;Br, 24.80. Found (percent): C, 56.09; H, 5.28; N, 4.59; Br, 25.05.

EXAMPLE 46 3-benzoyl-7-methoxy-3 -azabicyclo [3.3.1 ]nonan-6-one Asolution of 3-benzoyl-7-bromo-3-azabicyclo[3.2.2] nonan-6-one (1.888 g.,5.86 mmoles) in methanol was mixed with a solution of sodium hydroxide(0.485 g., 12.1 mmoles) in methanol (25 ml.). The solution was kept inthe dark at room temperature for 20 hours. The reaction solution wasconcentrated under reduced pressure to an oily solid, which was washedwith ethyl acetate. The ethyl acetate wash was placed on a column ofsilica gel (150 g.) packed with ethyl acetate, and eluted with ethylacetate. Fractions 6-8 were combined in acetone. Addition of SkellysolveB resulted in formation of crystals over a period of about 20 hours. Thecrystals (0.142 g.) thus obtained were collected and recrystallized fromacetone- Skellysolve B to give colorless crystals of 3-benzoyl-7-methoxy-3-azabicyclo[3.3.1]nonan-6-one, M.P. 114-116 C.; v 1710, 1625, v1600, 1490, v 775, 745, 700 cm.- in Nujol; NMR confirmed the assignedstructure.

Analysis.-Calcd. for C H NO (percent): C, 70.31; H, 7.01; N, 5.13. Found(percent): C, 70.08; H, 7.25; N, 5.39.

EXAMPLE 47 3-benzoyl-6- (N-pyrrolidyl) -3-azabicyclo [3.2.2] nonane,hydrochloride A mixture of 3-benzoyl-3-azabicyclo[3.2.2]nonan-6-one (11g.) 100 ml. of benzene, 16 ml. of pyrrolidine, and 200 mg. ofp-toluenesulfonic acid was stirred and heated at reflux under a watertrap for 3.5 hours and allowed to stand at 25 for 18 hours. The solventwas removed under reduced pressure on a hot water bath. The residualenamine was taken up in 100 ml. of ethanol and shaken with 1.0 g. of 10%palladium on carbon and hydrogen (40 p.s.i.g.) for 50 minutes. Thecatalyst was removed by filtration and the combined filtrate and washsolvent was concentrated under reduced pressure. The residue concentratethus obtained was taken up in 10% hydrochloric acid and extracted withether. The aqueous acid solution was made basic with 10% sodiumhydroxide solution and extracted with ether. This latter extract wasdried (Na SO and concentrated to give 14.6 g. of an oil. The oil waschromatographed over 500 g. of Florisil eluting with 7.8 l. of solvent.Skellysolve B containing increasing proportions of acetone from to 30%,and collecting fractions of 135 ml. each. Cuts 19-55 contained3-benzoyl-6-(N-pyrrolidyl)-3-azabicyclo[3.2.2]nonane as determined byTLC and IR; these were pooled in either, dried over sodium sulfate andtreated with ethereal hydrogen chloride to preciptate the aminehydrochloride, which was recovered, washed with ether, and crystallizedfrom acetone to yield 5.72 g. of3-benzoyl-6-(N-pyrrolidyl)-3-azabicyclo[3.2.2] nonane, hydrochloride,M.P. 228-230 C.

Analysis.Calcd. for C H N OCI (percent): C, 68.14; H, 8.13; N, 8.37; CI,10.59. Found (percent): C, 68.39; H, 8.38; N, 8.18; Cl, 10.37.

EXAMPLE 48 6- (N-pyrrolidyl) -3-azabicyclo 3.2.2] nonane dimaleate The3-benzoyl-6-(N pyrrolidyl) 3 azabicyclo[3.2.2] nonane obtained from 3.5g. of the corresponding hydrochloride salt obtained from Example 48,above, by treatment with aqueous sodium hydroxide and extraction withether and drying (Na SO was added to a stirred mixture of 3.0 g. ofLiAlH in 100 ml. of ether. The mixture was heated at reflux for twohours, chilled in a cold bath, and decomposed by the addition of 20 ml.of water. After dilution with more ether the mixture was filtered, andthe filtrate and ether wash were dried (MgSO This solution was made upto 400 ml. with ether and 300 ml. of this solution ether was taken todryness, the free base thus obtained was dissolved in 50 ml. of ethanoland shaken with 0.5 g. of 10% Pd on C and hydrogen (40 p.s.i.g.) for 17hours. The catalyst was removed by filtration and the solvent removedunder reduced pressure to yield6-(N-pyrrolidyl)-3-azabicyclo[3.2.2]nonane as an oil. This was made upto 100 ml. with ether. Ten milliliters of this solution was used toprecipitate the HCl salt which proved to be very hygroscopic. Twomilliliters of ether solution was used to prepare the p-toluenesulfonateby adding a few drops of ethanol solution of p-toluenesulfonic acid.

The remaining ether solution was treated with a methanolic solution ofmaleic acid to precipitate the maleate salt which was recovered andrecrystallized from methanol-methyl ethyl ketone to yield 1.54 g. of6-(N-pyrrolidyl)-3-azabicyclo[3.2.2]nonane dirnaleate, M.P. 167- 168' C.

Analyst's.-Calcd. for C H N O (percent): C, 56.32; H, 7.09; N, 6.57.Found (percent): C, 56.28; H, 7.66; N, 6.70.

EXAMPLE 49 6-methyl-3 -azabicyclo [3 .2.2] nonan-exo-G-ol, hydrochlorideTen grams of 3-benzoyl-3-azabicyclo[3.2.21nonan-exo- 6-01 in 200 ml. oftetrahydrofuran was added to a stirred solution of 100 ml. of 3 Mmethylmagnesium bromide in ether. The mixture was distilled until thevapor temperature was 60 and then heated at reflux for 4.5 hours. Thestirred mixture was chilled and treated with 60 ml. of water, followedby 50 ml. of acetic acid and extracted several times with ether. Theaqueous solution from the ether extraction was made basic with 50%sodium hydroxide and the resulting gelatinous mixture was continuouslyextracted with ether, the extract dried (M1 and treated with hydrogenchloride. The resulting HCl salt was recovered and washed with ether toyield 3.29 g. of 6- methyl-3-azabicyclo[3.2.2]nonan-exo-6-olhydrochloride, M.P. 228-230 C., which was recrystallized from methanolether, M.P. 230-232 C.

Analysis.-Calcd for C H NOCI (percent): C, 56.38; H, 9.46; N, 7.30; Cl,18.50. Found (percent): C, 56.67; H, 9.97; N, 8.09; Cl, 18.63.

EXAMPLE 50 6-phenyl-3-azabicyclo[3.2.2] nonan-exo-6-o1 hydrochloride Asolution of 20 g. of 3-benzoyl-3-azabicyclo[3.2.2] nonan-exo-6-ol in 400ml. of tetrahydrofuran was added to a stirred solution of 200 ml. of 3 Mphenylmagnesium bromide in ether. Solvent was removed by distillationuntil the boiling temperature was 60 and the mixture was heated atreflux for 4.5 hours. After cooling, it was poured onto ice and stirredand acidified with concentrated hydrochloride while continuing to addice. This mixture was extracted several times with ether. The aqueousacid solution was made basic with 50% sodium hydroxide solution and theresulting mixture was extracted several times with ether. The etherextract was dried (MgSO'g), treated with ethereal hydrogen chloride, andthe insoluble salt was recovered and washed with ether to yield 15.32 g.This was fractionally crystallized from methanol-methyl ethyl ketone togive about 7.5 g. of 6-phenyl-3-azabicyclo[3.2.2]nonan-exo-6-olhydrochloride, M.P. 238-240 C. (dec.).

Analysis.Calcd. for C H NOCI (percent): C, 66.26; H, 7.94; N, 5.52; Cl,13.97. Found (percent): C, 66.51; H, 8.17; N, 5.49; Cl, 13.90.

The filtrates from above produced about 2.0 g. of6-phenyl-3-azabicyclo{3.2.21nonan-endo 6 ol hydrochloride, M.P. 218-220C. (dec.).

Analysis.-Calcd. for C H NOCl (percent): C, 66.26; H, 7.94; N, 5.52; Cl,13.97. Found (percent): C, 66.09; H, 8.33; N, 5.36; Cl, 13.70.

29 EXAMPLE s1 1,4-dioxa-8-azaspiro[4.6]umdecane Asolution of1,4-dioxa-8-azaspiro[4.6]undecane (0.622 g., 0.00396 mole) in 16%aqueous sodium hydroxide was shaken with hydrogen and 5%palladium-on-carbon (2.50 g.) until the uptake of hydrogen ceased. Thecatalyst was removed by filtration and washed with methanol. Thecombined methanol solution was concentrated under reduced pressure to anoil. Simple distillation of the oil gave 5.047 g. (0.0321 mole, 91%yield) of 1,4-dioxa-8-azaspiro[4.6]undecane as a colorless oil, B.P.58'60 C. (0.05 mm.); 11 1.4885; v 3330 cm." neat.

Analysis.--Calcd. for C H NO (157.21) (percent): C, 61.12; H, 9.62; N,8.91. Found (percent): C, 61.17; H, 9.86; N, 8.85.

EXAMPLE 52 8-p-toluenesulfonyl-1,4dioxa-8-azaspiro [4.6]-

undecane A solution of 1,4-dioxa-8-azaspiro[4.61undecane (0.622 g.,0.00396 mole) in 16% aqueous sodium hydroxide was mixed withp-toluenesulfonyl chloride (0.7 86 g., 0.00413 mole). The mixture wasshaken vigorously for several minutes, then warmed and shaken more. Themixture was left at room temperature overnight and then was extractedwith ether (2X 20 ml.). The ether solution was dried and concentrated togive 8-p-toluenesulfonyl-1,4- dioxa-8-azaspiro[4.6]undecane as an oil.

EXAMPLE 5 3 1,4-dioxa-9-azaspiro [4.7] dodecane A solution of9-benzyl-1,4-dioxa-9 azaspiro[4.7]dode cane (24.642 g., 0.0945 mole) inabsolute ethanol (150 ml.) was shaken with 5% palladium-on-carbon (5.88g.) and hydrogen in a hydrogenation apparatus. After. 30 minutes thehydrogen uptake had stoppedand totaled 28 pounds (calculated, 27.4pounds). The catalyst was removed by filtration. The catalyst was washedtwice with ethanol. Ethanol was removed from the combined solvent andwashes by distillation. The residual oil was purified by a simpledistillation which gave 1,4-dioxa-9-azaspiro[4.7]dodecane as a colorlessoil (14.447 g., 0.0844 mole, 89%), RP. 70-75 C. (0.2 mm.); n 1.4835; v336O' cm. on the oil.

Analysis.Calcd. for C9H17NO2 (171.23) (percent): C, 63.13; H, 10.00; N,8.18. Found (percent): C, 63.31; H, 10.02; N, 8.17.

EXAMPLE 54 l,4-dioxa-8-azaspiro[4.7]dodecane A solution of8-benzyl-1,4-dioxa-8-azaspiro[4.7]dodecane (5.152 g., 0.0197 mole) inabsolute ethanol (75 ml.) was shaken with 5% palladium-on-carbon (1.5g.) and hydrogen in a hydrogenation apparatus for 3 hours. Hydrogenuptake was complete after one hour. The catalyst was removed byfiltration and was washed twice with methanol. The excess solvent wasremoved by distillation under reduced pressure. Distillation of theresidual oil gave 2.771 g. (0.0162 mole, 82% yield) of 1,4-dioxa-8-azaspiro-[4.7]dodecane as a colorless oil, B.P. 68-69 C. (0.1 mm.) n1.4853; v 3360 m.w. cm. on the oil.

Analysis.Calcd. for C H NO (171.23) (percent): C, 63.13; H, 10.00; N,8.18. Found (percent): C, 63.16; H, 10.04; N, 7.88.

EXAMPLE 55 1,4-dioxa-9-azaspiro [4.8]tridecane A solution of9-benzyl-1,4-dioxa-9-azaspiro[4.8']tridecane (9.451 g., 0.0344 mole) inmethanol (150 ml.) was shaken with 5% palladium-on-carbon (2.5 g.) inhydrogen for minutes at which time uptake of hydrogen appeared complete.The catalyst was removed by filtration and the colorless filtrate storedovernight in the refrigerator. The solution was concentrated underreduced pressure. The oil thus obtained crystallized as it cooled andthe solid was dissolved in ether, decolorized with activated charcoal,filtered and crystallized by addition of hexanes to the ether and bycooling in the freezer to give 4.649 g. ofl,4-dioxa-9'-azaspiro[4.8]tridecane (0.0251 mole, 73% yield), M.P. 55-57C.; recrystallization from ether-hexanes gave colorless, chunky crystalsof 1,4-dioxa-9-azaspiro[4.8]tridecane, M.P. 55- 57 C.; v 3400 m.w. cm?in Nujol.

Analysia-Calcd. forC H NO (185.26) (percent): C, 64.83; H, 10.34; N,7.56. Found (percent): C, 64.66; H, 10.40; N, 7.62.

EXAMPLE 56 3 -azabicyclo 3 .2 .2] nonan-6-one hydrochloride3-benzyl-3-azabicyclo[3.2.2]nonan-6-one (4.55 g.) was dissolved in ml.of ethanol and shaken with 1.0 g. of 10% palladium-on-carbon andhydrogen (50 p.s.i.g.) for 180 minutes. The catalyst was removed byfiltration; the filtrate and wash were concentrated in vacuo to a smallvolume, diluted with ether, and treated with ethereal HCl.

The hydrochloride of the product was recovered, washed with ether anddried; yield, 3.12 g. of 3-azabicyclo[3.2.2] nonan-6-one hydrochloride,M.P. 21 8-220 C. (dec.); a sample from methanol-ether melted at 227-229C. (dec.). 1

Analysis.-Calcd. for C H NO-HCl (percent): C, 54.70; H; 8.03; N, 7.98;Cl, 20.19. Found (percent): C, 54.22; -H, 8.14; N, 7.98; CI, 20.64.

EXAMPLE 57 3-azabicyclo[3.2.2]nonan-endo-6-ol hydrochloride A solutionof 9.17 g. of 3-benzyl-3-azabicyclo[3.2.2] nonan-endo-6-ol in ml. ofethanol was shaken with 1.0 g. of 10% palladium carbon and hydrogen (44p.s.i.g. starting pressure) for 20 hours. The mixture, freed of catalystand concentrated in vacuo gave 5.60 g. of3-azabicyclo[3.2.2]nonan-endo-6-ol. A portion of the free base wasdissolved in ether and treated with ethereal HCl to precipitate theamine hydrochloride, which was recrystallized from methanol-methyl ethylketone to give 3-azabicyclo[3.2.2]nonan-endo-6-o1 hydrochloride, M.P.280 C. (dec.).

Analysis.Calcd. for C H NOCl (percent): C, 54.07; H, 9.08; N, 7.88; C],19.96. Found (percent): C, 54.17; H, 9.05; N, 8.01; C1, 19.99.

EXAMPLE 5 8 1,4- dioxa-9-azaspiro [4.7 dodec-9-yl acetonitrile Asolution of 1,4 dioxa 9-azaspiro[4.7]dodecane (10.215 g., 0.0597 mole)in benzene (25 ml.) was added slowly to a stirred mixture ofchloroacetonitrile (6.0 g., 0.0795 mole) in benzene ml.) and anhydroussodium carbonate (4.0 g.). During the course of addition (15 minutes),the mixture was warmed to near the reflux temperatureand then was heatedto reflux with stirring for 18 hours. The precipitate thus obtained wasdissolved in dilute aqueous sodium bicarbonate solution. The benzenelayer was washed twice with water and dried over magnesium' sulfate.Concentration of the benzeneunder reduced pressure gave an oil whichcrystallized upon cooling. The crystalline material dissolved in hotSkellysolve B leaving a small amount of gummy, yellow residue and acolorless solution. Cooling gave colorless crystals (8.960 g.), M.P.77-78 C. A second crop, M.P. 7577 C. (1.726 g., total 10.686 g., 0.0508mole, 85% yield) was obtained from the concentrated filtrate.Recrystallization from Skellysolve B gave 1,4-dioxa-9-azaspiro [4.7]-dodec-9-yl acetonitrile as colorless needles, M.P. 78*79 C.; v 2220 cm.-in Nujol.

31 EXAMPLE 59 9-(2-aminoethyl)-1,4-dioxa-9-azaspiro- [4.7] dodecane Asolution of 1,4 dioxa 9-azaspiro[4.7]dodec-9-yl acetonitrile (8.095 g.,0.0385 mole) in ether (100 ml.) was added dropwise to a mixture oflithium aluminum hydride (5.0 g., 0.13 mole) in ether (200 ml.). Themixture was heated to reflux for three hours with stirring and then wasstirred at room temperature for 16 hours. The excess lithium aluminumhydride was decomposed with acetone-water and the inorganic saltsremoved by filtration. The solids were washed by warmin with ether (300ml.). The combined ether filtrates were dried over magnesium sulfate andconcentrated to an oil which was distilled, giving 7.074 g. (0.0330mole, 86% yield) of 9- (Z-aminoethyl)-1,4-dioXa-9 azaspiro[4.7]dodecaneas a colorless oil, B.P. 95101 C. (0.10 mm.); 11 1.4940; u 3360, 3280omf on the oil.

Analysis.Calcd. for C H N O (214.30) (percent): C, 61.65; H, 10.35; N,13.07. Found (percent): C, 61.28; H, 10.48; N, 13.22.

EXAMPLE 60 1-( 1 -guanidinylethyl --oxoheptamethyleneimine sulfateConcentrated sulfuric acid (1.17 g., 0.0120 mole) was added to asolution of 9-(2-aminoethyl)-1,4 dioxaspiro- [4.7]dodecane (5.116 g.,0.0239 mole) in water (20 ml). To this solution,2-methyl-2-thiopseudourea sulfate (3.34 g., 0.0120 mole) was added andthe resulting solution heated to reflux for 16 hours. Addition ofethanol to the cooled solution caused the slow formation of colorlesscrystals, first crop 4.129 g.; two additional crops formed upon additionof more ethanol, giving 1.595 g. (total 5.724 g., 0.0184 mole, 77%yield) of colorless crystals. The crops were combined and recrystallizedtwice from water-ethanol to give colorless crystals ofl-(l-guanidinylethyl)-5-oxoheptamethyleneimine sulfate, M.P. turningbrown to 300 C.; v 3200 m.s., 3070 s., .2640 m..w'., V N 1680 m.s., 1635m., 1600 m.w., cm." in Nujol.

Analysis.CalCd. for CH22N405S (p$r' cent): C, 38.69; H, 7.15; N, 18.05;S, 10.33. Found (percent): C, 38.82; H, 7.32; N, 18.07; S, 10.27.

EXAMPLE 61 3 -cyclohexyl-3 -azabicyclo 3 .2 .2] nonan-endo- 6-olhydrochloride .A mixture of 3.40 g. of 3-azabicyclo[3.2.2]nonan-endo6-01, 50 ml. of benzene, 3.0 ml. of cyclohexanone, and 7-0 mg. ofp-toluenesulfonic acid was stirred and heated at reflux for 2.5 hours.The mixture was concentrated to dryness under reduced pressure and theresidual oil was taken up in S -kellysolve B hexanes and filtered toremove insoluble material. The filtrate was concentrated under reducedpressure to an oil residue of enamine still containing cyclohexanone.This oil in 50 ml. of ethanol was shaken with 1.0 g. of 10% Pd on C andhydrogen (39 p.s.i.g.) for two hours. The catalyst was removed byfiltration and the filtrate concentrated under reduced pressure to givea crystalline residue of 3-cyclohexyl-3-azabicyclo[3.2.2]-endo-6-olwhich was dissolved in ether and treated with ethereal hydrogenchloride; the resulting HCl salt was recovered and recrystallized frommethanolmethyl ethyl 'ketone; yield, 3.43 g. of3-cyclohexyl-3-azabicyclo[3.2.2.]nonan-endo-6-ol, M.P. 265 C. (dec.).

Analysis.-Calcd. for C H NOCl (percent): C, 64.71; H, 10.01; N, 5.39;Cl, 13.65. Found (percent): C, 64.60; H, 10.21; N, 5.39; Cl, 13.74.

32 We claim: 1. The process for the introduction of oxygen into acompound of the formula:

wherein n is a whole number from 4 to 12, inclusive, X and X are eachselected from the group consisting of hydrogen, methyl, ethyl and propyland X and X taken together constitute a bridge containing from 1 to 3carbon atoms, inclusive, and Z is selected from the group consisting ofO O -(|-R,-SO2R and -g--OR in which R is an aryl substituent of 6 to 12carbon atoms, inclusive, and R( is an aralkyl substituent of 7 to 16carbon atoms, inclusive, which comprises subjecting said compound to theoxygenating activity of Sporotrichum sulfu'rescens.

2. The process of claim 1, wherein the oxygenation is carried out in anaqueous nutrient medium under submerged aerobic fermentation conditions.

3. The process of claim 1, wherein the oxygenation is carried out in anaqueous nutrient medium under submerged aerobic fermentation conditionsand the fermentation is continued until a substantial amount ofoxygenated products are produced and the oxygenated products thusobtained are recovered from the fermentation medium.

4. The process for the production of an oxygenated compound of theformula:

wherein n is a whole number from 1 to 5, inclusive, n is a whole numberfrom 2 to 8, inclusive, and in which the sum of in and n' is not lessthan 3 and not more than 11; X and X are each selected from the groupconsisting of methyl, ethyl, and propyl and X and X taken togetherconstitute a bridge containing from 1 to 3 carbon atoms, inclusive, andZ is selected from the group consisting of iJ-R, -'S02 R and (3OR1 inwhich R is an aryl substituent of 6 to 12 carbon atoms, inclusive, and Ris an aralkyl substituent of 7 to 16 carbon atoms, inclusive, whichcomprises subjecting a compound of the formula:

tag), 0 X1 wherein n is a whole number from 4 to 12, inclusive, and X,X; and Z are defined as above, to the oxygenating activity ofSporotrichum: sulfurescens.

S. The process of claim 4, wherein the oxygenation is carried out in anaqueous nutrient medium under submerged aerobic fermentation conditions.

6. The process of claim 4, wherein the oxygenation is carried out in anaqueous nutrient medium under submerged aerobic fermentation conditions,the fermentation is continued until a substantial amount of oxygenatedproducts is produced and the oxygenated products thus obtanied arerecovered from the fermentation medium.

7. The process of claim 4, wherein l-benzoylhexamethyleneimine issubjected to the oxygenating activity of Sporotrichum sulfurescens in anaqeous nutrient medium under aerobic fermentation conditions to obtain1-benzyo1-3 -oxohe'xamethyleneimine and1-benzoyl-4-oxohexamethyleneimine.

8. The process of claim 4, whereinl-(p-toluenesulfonyl-hexamethyleneimine is subjected to the oxygenatingactivity of Sporotrichum sulfurescens in an aqueous nutrient mediumunder aerobic fermentation conditions to obtain l- (p-toluenesulfonyl-4-hydroxyhexamethyleneimine.

9. The process of claim 4, wherein l-benzoylheptamethyleneimine issubjected to the oxygenating activity of Sporotrichum sulfurescens in anaqueous nutrient medium under aerobic fermentation conditions to obtain1- benzoy1-4-hydroxyheptamethyleneimine and 1-benz0y1-S-hydroxyheptamethyleneimine. I 10. The process of claim 4, whereinl-(p-toluenesulfonyl)-heptarnethyleneimine is subjected to theoxygenating activity of Sporotrichum sulfurescens in an aqueous nutrientmedium under aerobic fermentation conditions to obtainl-(p-toluenesulfonyl) 4 hydroxyheptamethyleneimine.

11. The process of claim 4, wherein l-benzoyloctamethyleneimine issubjected to the oxygenating activity of Sporatrichum sulfurescens in anaqueous nutrient medium under aerobic fermentation conditions to obtain1- benzoy1-4-hydroxyoctamethyleneimine and 1 benzoyl 5-hydroxyoctamethyleneimine.

12. The process of claim 4, wherein l-benzoyldodecamethyleneimine issubjected to the oxygenating activity of Sporotrichum sulfurescens in anaqueous nutrient medium under aerobic fermentation conditions to obtain1- benzoyl-S-hydroxydodecamethyleneimine, 1 benzoyl-6-hydroxydodecamethyleneimine, and1-benzoyl-7-hydroxydodecamethyleneimine.

13. The process of claim 4, wherein bispentamethyleneurea is subjectedto the oxygenating activity of Sporatrichum sulfures cens in an aqueousnutrient medium under aerobic fermentation conditions to obtainl-pentamethylenecarbamyl-4-hydroxypiperidene.

14. The process of claim 4, wherein 2-benzoyl-2- azabicyclo[2.2.2]octaneis subjected to the oxygenating activity of Sporotrichum sulfurescens inan aqueous nutrient medium under aerobic fermentation conditions toobtain Z-benzoyl-S-hydroxy-Z-azabicyclo[2.2.2]octane.

15. The process of claim 4, wherein 3-benzoyl-3-azabicyclo[3.2.2]nonaneis subjected to the oxygenating activity of Sporotrichum sulfurescens inan aqueous nutrient medium under aerobic fermentation conditions toobtain 3-benzoyl-3-azabicyclo[3.2.2]nonan-6-one and 3-benzoyl-3-azabicyclo 3 .2.2] nonan-6-ol.

16. The process of claim 4, wherein benzyl3-azabicyclo[3.2.2]nonane-3-carboxylate is subjected to the oxygenatingactivity of Sporotrz'cllum sulfurescens in an aqueous nutrient mediumunder aerobic conditions to obtain benzyl6-hydroxy-3-azabicyclo[3.2.2]nonane 3 carboxylate.

17. The process of claim 4, wherein 3-benzoyl-3-azabicyclo[3.2.l]nonaneis subjected to the oxygenating activity of Sporotrichum sulfurescens inan aqueous nutrient medium under aerobic fermentation conditions toobtain 3-benzoy1-3-azabicyclo [3 3 1 ]nonan-6-ol.

References Cited UNITED STATES PATENTS 3,392,171 7/1968 Fonken et a1 5lALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 5,55 ,93 Dated January 19, 1971 Inven fl Gunther S Fonken, Mi Iton E. Ifirr.Herbert C Mur It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

Col umn 29, I ine for "of 1,4-" read "of S-benzyl -1, Column 29, lines4-5, for "(0.622 9., 0.00596 mole) in 16% aqueous sod? um hydroxide wasshaken" read "(8.692 g O .0352 mole) in methanol (130 ml was shaken--Col umn 32, I ine 2 for "R(" read "R 81 ned and sealed this 22nd day ofJune 1971.

(SEAL) Attest:

EDWARD M.FLETCI-ER, JR. WILLIAM E. SCHUYLER, JR. Attesting OfficerCommissioner of Patents USCOMM-DC 60375 5 FORM PO-105O (10-69)

